Process for generating genetically engineered autologous t cells

ABSTRACT

The present invention relates to production of autologous genetically engineered T cells for use in cell therapy applications.

FIELD OF DISCLOSURE

The present invention relates to production of autologous geneticallyengineered T cells for use in cell therapy applications.

BACKGROUND

Adoptive T-cell therapy makes use of genetically engineered T cells andhas been shown to be an effective and powerful therapeutic treatment incertain hematological indications. Autologous T cells are geneticallyengineered to express one or more cell surface receptors, such aschimeric antigen receptors (CARs) or T-cell receptors (TCRs), thatrecognize proteins of interest associated with the surface of targetcells, while retaining and/or enhancing their ability to recognize andkill the target cells. In addition to the genetically engineered Tcells' ability to recognize and destroy the targeted cells, successfuladoptive T-cell therapy benefits from the T cells' ability to persist inthe patient and continue to proliferate in response to a target antigen.Genetically engineered autologous T cells have shown great success intreating a wide range of cancers.

For autologous T cell therapeutics, the product is a live cell from aspecific patient that will be returned to that patient. As such,autologous cell production is more challenging than conventionalbiopharmaceutical processes because one lot is manufactured for onepatient. Manufacturing can only start when a patient sample is receivedand care must be taken that the cells are returned to the patientwithout significant modifications that make the cells a risk rather thanan efficacious treatment. Production is more hands on and laborintensive, and performed multiple times at a small scale, to producecells at sufficient quantity and quality. Donor to donor variabilityincreases the complexity, because the starting material is different foreach new lot produced.

The specifications for assessing an autologous drug product are alsodifferent from a typical recombinant protein drug product. Forautologous T-cell therapeutics, product purity, cell phenotype, percentengineered T cells, potency and specificity, are the parameters tested.Integrated closed process platforms using complex automated systems arebeing developed but carry a high capital investment. Developing anefficient, scalable, reliable, and cost-effective process remains one ofthe main challenges for the advancement of use of autologous cells as atherapeutic, which is for the most part a highly manual process.

There is a need for production processes for generating T-celltherapeutics, particularly autologous T-cell therapeutics, that reducerisk of contamination, cost, facility footprint, and the inefficienciesand complexity of current manufacturing processes. Autologous T-celltherapies are forecast to be in the range of $350,000 to $475,000 perpatient, not including hospital-associated costs. If geneticallyengineered autologous T cells are to achieve their full clinical andcommercial potential, the challenges facing production of clinical gradecells at commercially relevant quantities and quality must be overcome.The invention described herein meets this need by providing an efficientand effective method manufacture to genetically engineered T cells forautologous cell therapy.

BRIEF SUMMARY OF THE INVENTION

The invention provides a method for producing genetically engineeredautologous T cells expressing at least one protein of interest, themethod comprising inoculating a closed single use bioreactor bagcontaining culture media with apheresed donor cells and one or moresoluble T cell activators, wherein the bioreactor bag is part of arocking bioreactor platform, culturing the cells in the closed singleuse bioreactor bag continuously rocking at a rate of about 2 RPM,transducing the cells in the closed single use bioreactor bag with atleast one soluble viral vector comprising a polynucleotide which encodesthe protein of interest continuously rocking at a rate of about 2 RPM,and expanding the cells in the closed single use bioreactor bag at arocking rate of about 2 RPM and increasing the culture volume androcking rate as needed to maintain the culture until harvest. In oneembodiment the apheresed donor cells comprise cells from peripheralblood. In a related embodiment the apheresed donor cells comprisenucleated and non-nucleated cells. In one embodiment the apheresed donorcells comprise leukocytes and erythrocytes. In a related embodiment theapheresed donor cells also comprise granulocytes and/or platelets. Inone embodiment the apheresis is leukapheresis. In one embodiment theapheresed donor cells are washed and resuspended in a culture media. Inone embodiment at least one T cell activator is an anti CD3 antibody orbinding fragments thereof. In yet another embodiment the T cellactivator comprises an anti CD3 antibody and an anti CD28 antibody, orbinding fragments thereof. In one embodiment the T cell activatorcomprises at least an anti CD3 antibody, an anti CD28 antibody, and ananti CD2 antibody, or binding fragments thereof. In another embodimentthe T cell activator comprises at least an anti-human CD3 monospecifictetrameric antibody complex, an anti-human CD28 monospecific tetramericantibody complex, and an anti-human CD2 monospecific tetrameric antibodycomplex. In another embodiment the concentration of at least one solubleT cell activator is at least 0.001 μg/ml to at least 10 μg/ml. In arelated embodiment the concentration of at least one soluble T cellactivator is at least 0.1 μg/ml to at least 5 μg/ml. In yet anotherembodiment at least one soluble T-cell activator is bound to at leastone donor cell at the time of inoculation. In another embodiment theapheresed donor cells are incubated with one or more soluble T cellactivators prior to inoculating into the bioreactor bag. In a relatedembodiment the incubation is for a sufficient time to allow forsaturation of binding of one or more soluble T cell activator to theapheresed donor cells prior to inoculation. In a related embodiment theapheresed donor cells and one or more soluble T cell activators areincubated in a transfer bag. In a related embodiment the volume ofculture media in the transfer bag is about 5 ml to about 50 ml. In arelated embodiment the volume of culture media in the transfer bag isabout 5 ml to about 10 ml. In a related embodiment the apheresed donorcells are incubated with one or more T cell activators for at least 30minutes or more. In a related embodiment the apheresed donor cells areincubated with one or more T cell activators for at least 1 hour. In oneembodiment the number of nucleated cells within the apheresed donorcells is about 1.0E9 to about 1.3E9. In one embodiment the number ofnucleated cells within the apheresed donor cells is about 1.2E9. In oneembodiment the bioreactor bag is inoculated with apheresed donor cellsat a cell density of about 1E6 to about 5E6 nucleated cells/ml. In arelated embodiment the bioreactor bag is inoculated with apheresed donorcells at a cell density of about 2E6. In one embodiment the bioreactorbag contains at least 300 ml to at least 400 ml of culture media atinoculation. In a related embodiment the bioreactor bag contains atleast 300 ml of culture media at inoculation. In one embodiment theapheresed donor cells are cultured in the bioreactor bag for about 12-24hours. In one embodiment the culture media comprises at least onesoluble cytokine. In a related embodiment the soluble cytokine selectedfrom IL-2, IL-7, IL-15, or IL-21. In a related embodiment at least onesoluble cytokine is IL-2. In a related embodiment the IL-2 is at aconcentration of about 250 IU/ml to about 350 IU/ml. In a relatedembodiment the IL-2 is at a concentration of about 300 IU/ml. In arelated embodiment the soluble cytokine is IL-7 in combination withIL-15 or IL-21. In a related embodiment the concentration of at leastone cytokine is at least 5 ng/ml to at least 30 ng/ml. In a relatedembodiment the concentration of at least one cytokine is at least 10ng/ml to at least 20 ng/ml. In one embodiment the culture media alsocomprises a WNT pathway activator. In a related embodiment the WNTpathway activator is TWS117. In a related embodiment the culture mediacomprises a mixture of soluble TWS117, IL-7, and IL-21. In oneembodiment the culture media also comprises a soluble glycolysisinhibitor. In a related embodiment the soluble glycolysis inhibitor is2-deoxy-D-glucose (2-DG). In one embodiment the viral vector is aretroviral vector. In one embodiment the viral vector is a lentiviralvector. In one embodiment the lentiviral vector is added at a MOI of0.25-10. In a related embodiment the lentiviral vector is added at a MOIof 1. In one embodiment the cells are transduced for about 20-24 hours.In one embodiment following transduction, half of the culture media isremoved from the bioreactor bag and replaced with an equal volume offresh culture media. In a related embodiment the culture is incubatedfor about 12-24 hours. In one embodiment during expansion, fresh culturemedia is added to the bioreactor by fed batch/perfusion feeding and/orby perfusion. In one embodiment during expansion the culture is perfusedat a rate is one bioreactor bag volume per day. In one embodiment thecells are expanded the volume of the culture media in bioreactor isincrementally increased to 1 liter during expansion. In one embodimentas the cells are expanded the volume of the culture media isincrementally increased to maintain a cell density of at least 2E6nucleated cells/ml. In one embodiment as the cells are expanded thevolume of the culture media in bioreactor is incrementally increased to1 liter during expansion to maintain a cell density of at least 4E6nucleated cells/ml. In one embodiment as the cells are expanded therocking rate is incrementally increased to 6 RPM. In one embodiment atthe start of expansion, the volume of culture media in the single useclosed bioreactor bag is 300 ml rocking at a rate of 2 rpm at a 2°angle. In one embodiment the culture in the single use closed bioreactorbag is maintained at about 80-100% O₂. In one embodiment the cells areexpanded for 7 to 14 days. In one embodiment the culture, transduction,and/or expansion steps are performed at 34-37° C. In one embodiment theprotein of interest is a cell surface receptor. In a related embodimentthe cell surface receptor a T cell receptor, or chimeric antigenreceptor. In a related embodiment the cell surface receptor recognizesan antigenic target associated with a target cell. In a relatedembodiment the target cell is a cancer cell. In one embodiment thegenetically engineered autologous T cells are used to treat anindication in a patient in need.

The invention provides a pharmaceutical composition comprising thegenetically engineered autologous T cells made using the methoddescribed above.

The invention provides a method of treating an indication in a patientin need, comprising administering to the patient the pharmaceuticalcomposition as described above.

The invention provides a method for increasing the transgene expressionin genetically engineered autologous T cells expressing a protein ofinterest, the method comprising inoculating a closed single usebioreactor bag containing culture media with apheresed donor cells andone or more soluble T cell activators, wherein at least one solubleT-cell activator is bound to at least one donor cell at the time ofinoculation and the bioreactor bag is part of a rocking bioreactorplatform, culturing the cells in the closed single use bioreactor bagcontinuously rocking at a rate of about 2 RPM, transducing the cells inthe closed single use bioreactor bag with at least one soluble viralvector comprising a polynucleotide which encodes the protein of interestcontinuously rocking at a rate of about 2 RPM, and expanding the cellsin the closed single use bioreactor bag at a rocking rate of about 2 RPMand increasing the culture volume and rocking the rate as needed tomaintain the culture until harvest, wherein the transgene expression isgreater than the transgene expression of genetically engineeredautologous T cells derived from an enriched population of T cells fromthe same apheresed donor cells and expressing the same protein ofinterest. In one embodiment the apheresed donor cells comprise cellsfrom peripheral blood. In a related embodiment the apheresed donor cellscomprise nucleated and non-nucleated cells. In one embodiment theapheresed donor cells comprise leukocytes and erythrocytes. In a relatedembodiment the apheresed donor cells also comprise granulocytes and/orplatelets. In one embodiment the apheresis is leukapheresis. In oneembodiment the apheresed donor cells are washed and resuspended in aculture media. In one embodiment at least one T cell activator is ananti CD3 antibody or binding fragments thereof. In yet anotherembodiment the T cell activator comprises an anti CD3 antibody and ananti CD28 antibody, or binding fragments thereof. In one embodiment theT cell activator comprises at least an anti CD3 antibody, an anti CD28antibody, and an anti CD2 antibody, or binding fragments thereof. Inanother embodiment the T cell activator comprises at least an anti-humanCD3 monospecific tetrameric antibody complex, an anti-human CD28monospecific tetrameric antibody complex, and an anti-human CD2monospecific tetrameric antibody complex. In another embodiment theconcentration of at least one soluble T cell activator is at least 0.001μg/ml to at least 10 μg/ml. In a related embodiment the concentration ofat least one soluble T cell activator is at least 0.1 μg/ml to at least5 μg/ml. In another embodiment the apheresed donor cells are incubatedwith one or more soluble T cell activators prior to inoculating into thebioreactor bag. In a related embodiment the incubation is for asufficient time to allow for saturation of binding of one or moresoluble T cell activator to the apheresed donor cells prior toinoculation. In a related embodiment the apheresed donor cells and oneor more soluble T cell activators are incubated in a transfer bag. In arelated embodiment the volume of culture media in the transfer bag isabout 5 ml to about 50 ml. In a related embodiment the volume of culturemedia in the transfer bag is about 5 ml to about 10 ml. In a relatedembodiment the apheresed donor cells are incubated with one or more Tcell activators for at least 30 minutes or more. In a related embodimentthe apheresed donor cells are incubated with one or more T cellactivators for at least 1 hour. In one embodiment the number ofnucleated cells within the apheresed donor cells is about 1.0E9 to about1.3E9. In one embodiment the number of nucleated cells within theapheresed donor cells is about 1.2E9. In one embodiment the bioreactorbag is inoculated with apheresed donor cells at a cell density of about1E6 to about 5E6 nucleated cells/ml. In a related embodiment thebioreactor bag is inoculated with apheresed donor cells at a celldensity of about 2E6. In one embodiment the bioreactor bag contains atleast 300 ml to at least 400 ml of culture media at inoculation. In arelated embodiment the bioreactor bag contains at least 300 ml ofculture media at inoculation. In one embodiment the apheresed donorcells are cultured in the bioreactor bag for about 12-24 hours. In oneembodiment the culture media comprises at least one soluble cytokine. Ina related embodiment the soluble cytokine selected from IL-2, IL-7,IL-15, or IL-21. In a related embodiment at least one soluble cytokineis IL-2. In a related embodiment the IL-2 is at a concentration of about250 IU/ml to about 350 IU/ml. In a related embodiment the IL-2 is at aconcentration of about 300 IU/ml. In a related embodiment the solublecytokine is IL-7 in combination with IL-15 or IL-21. In a relatedembodiment the concentration of at least one cytokine is at least 5ng/ml to at least 30 ng/ml. In a related embodiment the concentration ofat least one cytokine is at least 10 ng/ml to at least 20 ng/ml. In oneembodiment the culture media also comprises a WNT pathway activator. Ina related embodiment the WNT pathway activator is TWS117. In a relatedembodiment the culture media comprises a mixture of soluble TWS117,IL-7, and IL-21. In one embodiment the culture media also comprises asoluble glycolysis inhibitor. In a related embodiment the solubleglycolysis inhibitor is 2-deoxy-D-glucose (2-DG). In one embodiment theviral vector is a retroviral vector. In one embodiment the viral vectoris a lentiviral vector. In one embodiment the lentiviral vector is addedat a MOI of 0.25-10. In a related embodiment the lentiviral vector isadded at a MOI of 1. In one embodiment the cells are transduced forabout 20-24 hours. In one embodiment following transduction, half of theculture media is removed from the bioreactor bag and replaced with anequal volume of fresh culture media. In a related embodiment the cultureis incubated for about 12-24 hours. In one embodiment during expansion,fresh culture media is added to the bioreactor by fed batch/perfusionfeeding and/or by perfusion. In one embodiment during expansion theculture is perfused at a rate is one bioreactor bag volume per day. Inone embodiment the cells are expanded the volume of the culture media inbioreactor is incrementally increased to 1 liter during expansion. Inone embodiment as the cells are expanded the volume of the culture mediais incrementally increased to maintain a cell density of at least 2E6nucleated cells/ml. In one embodiment as the cells are expanded thevolume of the culture media in bioreactor is incrementally increased to1 liter during expansion to maintain a cell density of at least 4E6nucleated cells/ml. In one embodiment as the cells are expanded therocking rate is incrementally increased to 6 RPM. In one embodiment atthe start of expansion, the volume of culture media in the single useclosed bioreactor bag is 300 ml rocking at a rate of 2 rpm at a 2°angle. In one embodiment the culture in the single use closed bioreactorbag is maintained at about 80-100% O₂. In one embodiment the cells areexpanded for 7 to 14 days. In one embodiment the culture, transduction,and/or expansion steps are performed at 34-37° C. In one embodiment theprotein of interest is a cell surface receptor. In a related embodimentthe cell surface receptor a T cell receptor, or chimeric antigenreceptor. In a related embodiment the cell surface receptor recognizesan antigenic target associated with a target cell. In a relatedembodiment the target cell is a cancer cell. In one embodiment thegenetically engineered autologous T cells are used to treat anindication in a patient in need.

The invention provides a method of treating a patient with geneticallyengineered autologous T cells expressing a protein of interestcomprising, incubating apheresed cells from the patient with one or moreT cell activators selected from the group consisting of an anti CD3antibody, an anti CD2 antibody, and an anti CD28 antibody or bindingfragments thereof, to allow for saturation of antibody binding,inoculating a closed single use bioreactor bag containing culture mediawith the apheresed cells, wherein the bioreactor bag is part of arocking bioreactor platform, culturing the cells in the closed singleuse bioreactor bag continuously rocking at a rate of about 2 RPM,transducing the cells in the closed single use bioreactor bag with atleast one soluble viral vector comprising a polynucleotide which encodesthe protein of interest continuously rocking at a rate of about 2 RPM,and expanding the cells in the closed single use bioreactor bag at arocking rate of about 2 RPM, increasing the culture volume and rockingthe rate as needed to maintain the culture at a desired cell densityuntil harvest, harvesting and formulating the cells forcryopreservation, freezing the cells and storing until needed foradministering to the patient, thawing and resuspending the cells in asuitable media for infusion, and reintroducing a pharmaceuticallyeffective amount of the genetically engineered autologous T cellsexpressing the protein of interest into the patient. In one embodimentthe apheresed donor cells comprise cells from peripheral blood. In arelated embodiment the apheresed donor cells comprise nucleated andnon-nucleated cells. In one embodiment the apheresed donor cellscomprise leukocytes and erythrocytes. In a related embodiment theapheresed donor cells also comprise granulocytes and/or platelets. Inone embodiment the apheresis is leukapheresis. In one embodiment theapheresed donor cells are washed and resuspended in a culture media. Inone embodiment at least one T cell activator is an anti CD3 antibody orbinding fragments thereof. In yet another embodiment the T cellactivator comprises an anti CD3 antibody and an anti CD28 antibody, orbinding fragments thereof. In one embodiment the T cell activatorcomprises at least an anti CD3 antibody, an anti CD28 antibody, and ananti CD2 antibody, or binding fragments thereof. In another embodimentthe T cell activator comprises at least an anti-human CD3 monospecifictetrameric antibody complex, an anti-human CD28 monospecific tetramericantibody complex, and an anti-human CD2 monospecific tetrameric antibodycomplex. In another embodiment the concentration of at least one solubleT cell activator is at least 0.001 μg/ml to at least 10 μg/ml. In arelated embodiment the concentration of at least one soluble T cellactivator is at least 0.1 μg/ml to at least 5 μg/ml. In yet anotherembodiment at least one soluble T-cell activator is bound to at leastone donor cell at the time of inoculation. In a related embodiment theincubation is for a sufficient time to allow for saturation of bindingof one or more soluble T cell activator to the apheresed donor cellsprior to inoculation. In a related embodiment the apheresed donor cellsand one or more soluble T cell activators are incubated in a transferbag. In a related embodiment the volume of culture media in the transferbag is about 5 ml to about 50 ml. In a related embodiment the volume ofculture media in the transfer bag is about 5 ml to about 10 ml. In arelated embodiment the apheresed donor cells are incubated with one ormore T cell activators for at least 30 minutes or more. In a relatedembodiment the apheresed donor cells are incubated with one or more Tcell activators for at least 1 hour. In one embodiment the number ofnucleated cells within the apheresed donor cells is about 1.0E9 to about1.3E9. In one embodiment the number of nucleated cells within theapheresed donor cells is about 1.2E9. In one embodiment the bioreactorbag is inoculated with apheresed donor cells at a cell density of about1E6 to about 5E6 nucleated cells/ml. In a related embodiment thebioreactor bag is inoculated with apheresed donor cells at a celldensity of about 2E6. In one embodiment the bioreactor bag contains atleast 300 ml to at least 400 ml of culture media at inoculation. In arelated embodiment the bioreactor bag contains at least 300 ml ofculture media at inoculation. In one embodiment the apheresed donorcells are cultured in the bioreactor bag for about 12-24 hours. In oneembodiment the culture media comprises at least one soluble cytokine. Ina related embodiment the soluble cytokine selected from IL-2, IL-7,IL-15, or IL-21. In a related embodiment at least one soluble cytokineis IL-2. In a related embodiment the IL-2 is at a concentration of about250 IU/ml to about 350 IU/ml. In a related embodiment the IL-2 is at aconcentration of about 300 IU/ml. In a related embodiment the solublecytokine is IL-7 in combination with IL-15 or IL-21. In a relatedembodiment the concentration of at least one cytokine is at least 5ng/ml to at least 30 ng/ml. In a related embodiment the concentration ofat least one cytokine is at least 10 ng/ml to at least 20 ng/ml. In oneembodiment the culture media also comprises a WNT pathway activator. Ina related embodiment the WNT pathway activator is TWS117. In a relatedembodiment the culture media comprises a mixture of soluble TWS117,IL-7, and IL-21. In one embodiment the culture media also comprises asoluble glycolysis inhibitor. In a related embodiment the solubleglycolysis inhibitor is 2-deoxy-D-glucose (2-DG). In one embodiment theviral vector is a retroviral vector. In one embodiment the viral vectoris a lentiviral vector. In one embodiment the lentiviral vector is addedat a MOI of 0.25-10. In a related embodiment the lentiviral vector isadded at a MOI of 1. In one embodiment the cells are transduced forabout 20-24 hours. In one embodiment following transduction, half of theculture media is removed from the bioreactor bag and replaced with anequal volume of fresh culture media. In a related embodiment the cultureis incubated for about 12-24 hours. In one embodiment during expansion,fresh culture media is added to the bioreactor by fed batch/perfusionfeeding and/or by perfusion. In one embodiment during expansion theculture is perfused at a rate is one bioreactor bag volume per day. Inone embodiment the cells are expanded the volume of the culture media inbioreactor is incrementally increased to 1 liter during expansion. Inone embodiment as the cells are expanded the volume of the culture mediais incrementally increased to maintain a cell density of at least 2E6nucleated cells/ml. In one embodiment as the cells are expanded thevolume of the culture media in bioreactor is incrementally increased to1 liter during expansion to maintain a cell density of at least 4E6nucleated cells/ml. In one embodiment as the cells are expanded therocking rate is incrementally increased to 6 RPM. In one embodiment atthe start of expansion, the volume of culture media in the single useclosed bioreactor bag is 300 ml rocking at a rate of 2 rpm at a 2°angle. In one embodiment the culture in the single use closed bioreactorbag is maintained at about 80-100% O₂. In one embodiment the cells areexpanded for 7 to 14 days. In one embodiment the culture, transduction,and/or expansion steps are performed at 34-37° C. In one embodiment theprotein of interest is a cell surface receptor. In a related embodimentthe cell surface receptor a T cell receptor, or chimeric antigenreceptor. In a related embodiment the cell surface receptor recognizesan antigenic target associated with a target cell. In a relatedembodiment the target cell is a cancer cell. In one embodiment thegenetically engineered autologous T cells are used to treat anindication in a patient in need.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. (A) Growth curve of cell expansion in Xuri W25 bioreactorsupplemented with IL2 only or cocktail of IL7, IL21, and TWS119. (B)Viability of cell expanded in Xuri W25 bioreactor supplemented with IL2only or IL7, IL21, and TWS119 cocktail.

FIG. 2. T cell transgene expression from engineered T cells expressed inthe three systems: Xuri W25 bioreactor (“Xuri”), combination of staticperfusion bag and Xuri W25 bioreactor (“Permeable Bag”) and G-Rex®plates (“G-Rex”).

FIG. 3. Leukocyte subsets in harvested engineered T cells expressed inthe three systems: Xuri W25 bioreactor (“Xuri”), combination of staticperfusion bag and Xuri W25 bioreactor (“Permeable Bag”) and G-Rex plates(“G-Rex”).

FIG. 4. Percentage of T cells subsets on Day 5 (A) and Day 10 (B) fromengineered T cells expressed in the three systems: Xuri W25 bioreactor(“Xuri”), combination of static perfusion bag and Xuri W25 bioreactor(“Permeable Bag”) and G-Rex plates (“G-Rex”).

FIG. 5. Shows the cytotoxic function of engineered T cells characterizedby their ability in target cell lysis (A), IFN-gamma release in responseto target cells (B), and TNF-alpha release in response to target cells(C).

FIG. 6. Resistance to target cell challenge of engineered T cellsexpressed in the three systems: Xuri W25 bioreactor (“Xuri”),combination of static perfusion bag and Xuri W25 bioreactor (“PermeableBag”) and G-Rex plates (“G-Rex”). Annexin level (A), Tim3 (B).

FIG. 7. Growth curve (A) and viability (B) for cells expanded in mediasupplemented with 2-DG (Media 1) and without 2-DG (Media 2).

FIG. 8. Transgene expression of cells expanded in the media supplementedwith 2-DG (Media 1) and without 2-DG (Media 2).

FIG. 9. CD25 expression by cells expanded in the media supplemented with2-DG (Media 1) and without 2-DG (Media 2).

FIG. 10. Glucose (A) and lactate (B) concentrations in culturessupplemented with 2-DG (Media 1) and without 2-DG (Media 2).

FIG. 11. T differentiation phenotyping of cells expanded mediasupplemented with (Media 1) and without 2-DG (Media 2).

FIG. 12. Leukocyte subsets in harvested T cells of cells expanded mediasupplemented with (Media 1) and without 2-DG (Media 2).

FIG. 13. Results from potency assays of engineered TCR T cells. (A)Cytotoxicity. (B) IFN-γ. (C) T cell differentiation.

FIG. 14. Transgene expression of T cells at harvest from enriched- andunenriched-process, gated on CD8+ cells (A) or CD4+ cells (B).

FIG. 15. Vector copy number of TCR in CD4⁺ and CD8⁺ T cells.

FIG. 16. Percentage of leukocytes and leukocyte subsets measured at theend of harvest.

FIG. 17. T cell phenotype at harvest from the enriched- andunenriched-cells from three donors.

FIG. 18. Growth curve (A) and viability (B) of cells in the bioreactorfrom enriched- and unenriched-donor cells.

FIG. 19. Growth curve and viability of cells.

FIG. 20. Percentage of leukocyte subsets at Day 0 and Day 10.

FIG. 21. T cell differentiation during bioprocessing.

FIG. 22 shows the expression of transgene in harvested T cells at cellsurface level (A) and DNA level (B).

FIG. 23. Percentage of killing of target cells at different effector totarget cells (E: T) ratios.

FIG. 24. IFN-gamma release of harvested cells in response to targetcells.

FIG. 25. Results of the titration of coated anti-CD3 (OKT3) antibody andpan T cell activation measured by CD69, CD35, and 4-1BB.

FIG. 26. Results of stimulation of pan T cells with various solubleactivators versus coated anti-CD3 as measured by CD69, CD25, and 4-1BB.

FIG. 27. Transduction efficiency for the engineered T cells, Pan T cells(A) and apheresed donor cells (B).

FIG. 28. Cell growth of cells activated by various activators.

FIG. 29. Effect of culture conditions on transduction efficiency.

FIG. 30. Cell density and transduction efficiency.

FIG. 31. Multiplicity of infection for TCR transduction. (A) MOI of1-10, (B) MOI of 0.25 to 2.

DETAILED DESCRIPTION OF THE INVENTION

Autologous T-cell therapy is a personalized therapy, the geneticallyengineered T cells being derived from the patient's own cells,“autologous cells”, which are manipulated and returned to the patient. Tcell or T lymphocyte refers to a type of lymphocyte (others are B cellsand NK cells) that actively participates in the body's immune response.While use of autologous cells reduces the risk of immunologicalresponses, manufacturing a therapy at one lot per patient addscomplexity and cost. Also, because autologous T-cell therapy ispatient-centric, this treatment is unlike traditional biologictherapeutics, and is not suitable for large scale production andoff-the-shelf use.

Autologous T-cell therapy involves treating patients with their own liveT cells that have been engineered to express a protein of interest thatrecognizes a protein associated with a target cell. Autologous T cellmanufacturing processes typically make use of multiple unit operationsincluding semi-closed and/or closed steps, moving the patient's cellsbetween multiple vessels such as plates, flasks, containers, bags, orother types of vessels, typically gas permeable vessels, over the courseof production. These transfers may involve movement of fluid from onevessel to another by means such as syringes or pipettes, centrifuges,and the like that can put unnecessary stress on the cells through shearforces and expose the cells to contaminants and loss. In addition, suchvessels require manual transfer into and between equipment, such as cellseparators, biosafety cabinets, and incubators.

As described herein, a method for producing genetically engineeredautologous T cells that express a protein of interest has been developedthat produces T cells of high purity, greater than 98%, with hightransgene expression, greater than 45% on CD8 T cells, generates cellyields of greater than 10 billion with a viability of greater than 90%in a 10-day completely closed and continuous process. Starting with anapheresed donor sample, the T cells are selectively expanded from about50% when harvested to greater than 98% following genetic manipulationand expansion of the activated cells. The resulting T cells are lessdifferentiated and remain in a more stem-cell or memory-like phenotypewhich increases persistence and over all efficacy. The resulting cellsare more potent in killing target cells and resistant target cellchallenge.

While the growth rate and viability were equivalent to cells that wereenriched prior to activation and transduction, the transduction was moreefficient, and the function and efficacy of the cells produced directlyfrom harvested donor cells were greater.

The genetically engineered autologous T cells were generated in a closedcontinuous rocking culture through the steps of activation,transduction, and expansion, using bioreactor capable of producing arocking motion throughout. The rocking was tailored to the variances inbioreactor volume, (increasing to 1 liter by the end of expansion), andoxygen levels as the cell density increased during the process. Thisensured a sufficient mass transfer of oxygen and nutrients to supportthe high-density cultures in contrast to methods using static culturewhich must be grown at low cell density (≤2E6 cells/ml) due tolimitations of the culture media under static conditions. While thecells that were rocked in a bioreactor bag and a commonly used staticG-Rex® gas permeable culture system both had efficient oxygen transport,the G-Rex® system lacked the convection flow that transfers nutrients tocells in a timely manner. Also, the bioreactor bags can take advantageof perfusion of fresh media which supports very high cell densities, upto 50E6 cells/ml in some cases.

Compared to cells generated in static, gas permeable vessels or culturesgenerated using a combination of static (activation and transductionoperations) and rocking (expansion operations) systems, the methoddescribed herein eliminated more than 30% of the hands-on work andgreatly reduced the cost. Incubators and other support equipment that isessential for preparing and maintaining static cultures in gas permeablevessels are not necessary, reducing the manufacturing footprint and riskof contamination of the cells and the equipment from the necessaryhandling and manipulation that these static cultures require. While thegrowth rate and viability of the cells generated by these systems wereequivalent, the transgene expression, function and efficacy of the cellsgenerated by the method described herein was greater than for the cellsgenerated in cultures making use of gas permeable vessels in static orhybrid static/rocking cultures.

The donor may be any subject from which a sample of blood cells isneeded for producing genetically engineered autologous T cells. Thedonor may be a patient in need of treatment with a population of cellsgenerated by the method described herein (i.e., an autologous donor).

The donor cells may be harvested from the circulating peripheral bloodby any suitable method used in the art such as extracorporeal methods,venipuncture, or other blood collection methods by which a sample ofblood is typically obtained. In one embodiment, the donor cells areharvested by apheresis. Apheresis refers to the withdrawal of blood froma donor, which is separated into cellular and soluble components,removing certain desired components from the blood and then returningthe remainder of the blood to the donor. Where T cells are a desiredcell type, such as for cell therapy applications, leukapheresis, anapheresis process which preferentially removes white blood cells(leukocytes) from the peripheral blood of a donor, is most often used.The harvested leukapheresis sample may be provided in a Leukopak®container. Multiple blood volumes are processed from the same donor togenerate a full Leukopak®.

Apheresis separates the incoming blood into the various blood componentsusing methods such as differential centrifugation. However, becausethere is a close range in the density between blood components, thiswill not result in pure cell populations, there will be residual cellsin any sample collected by an apheresis process, and the quantities ofthese residual cells will increase as the volume of blood processedduring the apheresis procedure increases. Cells harvested byleukapheresis will comprise nucleated cells, such as leukocytes,including monocytes, dendritic cells, lymphocytes (T-, B-, and NKcells), and granulocytes, as well as megakaryocytes, and cells without anucleus, such as erythrocytes and platelets. While the percentage oflymphocytes in the collected apheresed sample is more concentratedcompared to whole blood, there will also be a significant number ofother cells such as NK cells, red blood cells, platelets, andgranulocytes in the sample as well.

Apheresed cells are frequently used as source for collecting blood-basedcells, such as leukocytes and lymphocytes. However, since these samplesinclude a mixture of blood cells, for example, Leukopack®s containingapheresed cells can contain tens of billions of red blood cells makingthe collected cell sample visibly red. Red blood cells are known to havean impact further processing of the cells in the sample, such as bydrastically lower transfection rates when using electroporation, forexample. “As there is inherent variability in the cell populations inthese leukapheresis products, processes to remove unwanted cells orisolate specific populations of cells have been developed using avariety of technologies including physical separation viacentrifugation, magnetic, fluorescent, as well as acoustic-basedselection. Cells types can be separated based on size throughcentrifugation, with or without the use of density gradient mediasystems (such as Ficoll, for example), which enables removal of unwantedfractions of leukapheresis product such as granulocytes, platelets andremaining red blood cell contaminants.” Iyer et al., (2018) Frontiers inMedicine, Vol. 5, 150. It is common practice in production of autologousand allogenic T cells to further isolate, select, and/or enrich fordesired cell populations or phenotypes from the apheresed sample beforeusing the cells in a particular method or application related to celltherapy. The purpose of this further processing is to obtain a morehighly defined population of cells, such as PBMCs, or specificlymphocyte populations, for uses such as T cell activation or expansion,see for example, Stroncek et al. Journal of Translational Medicine 2014,12:241-249.

Procedures for obtaining desired cell populations, such as enrichment ofcertain T cell phenotypes, are one of the most expensive unit operationsin an autologous T cell manufacturing process, averaging around $10K perpatient in a clinical setting using the CliniMACs Plus magneticseparation technique, for example. Even after enrichment, T cell purityonly reaches 90-95% and some 10% of T cells may be lost in the negativefraction (flow-through).

The standard procedure for separation of red blood cells andgranulocytes from the PBMCs and lymphocytes in a harvested blood sample,such as apheresed cells, is by use of density gradient centrifugation.The typical density gradient material used for this purpose is Ficoll®,a hydrophilic polysaccharide, that separates the components in bloodsamples, such as whole blood or blood collected via an apheresis processor other method. A Ficoll® density gradient separates mononuclear cells,dendritic cells and lymphocytes (T, B and NK cells) which are found inthe buffy coat and separated from the red blood cells, platelets, andgranulocytes which are found in the pellet. However, different Ficoll®separation media, protocols, operator skill, and the like, can affectthe yield, viability, and function of the cells. In addition, in someprocesses the cells are manipulated in a hyperosmolar Ficoll® solutionfollowed by resuspension in artificial media, which may have profoundmodifying effects on T cell function, see Mallone et al., Clinical andExperimental Immunology, 163: 33-49 (2010). Also, lymphocytes expressinghigh-avidity binding for autologous erythrocytes have been shown to leadto a loss of these T-rosetting cells during Ficoll® separation, see forexample, Hokland et al. Scand. J. Immunol, 11, 353-356 (1980).

In addition, apheresed cells that are further isolated, selected and/orenhanced are subjected to additional manipulations, such ascentrifugation as part of wash and separation processes, densitygradients, and the like. This can lead to cell damage and loss due tothe repeated shear forces experienced during these processes. Inaddition, each time the cells are handled, whether manually or as partof an automated process, there is an increased risk of exposure andcontamination of the cells. For a manufacturing facility where a highvolume of samples from different donors are processed for use inautologous cell therapy applications, contamination of a processedsample with foreign donor cells is a serious risk and danger to thedonor patient.

The method described herein makes use of the entire apheresed samplewithout further isolation, separation, and/or enrichment for specificcell populations or phenotypes. The method starts with billions of cellsharvested by leukapheresis which comprise nucleated cells, such asleukocytes, including monocytes, dendritic cells, lymphocytes (T-, B-,and NK cells), and granulocytes, as well as megakaryocytes, and cellswithout a nucleus, such as erythrocytes and platelets. While only about30-60% of the cells in the apheresed sample are T cells, the othersupporting cells have a positive effect on the outcome of the method,contributing to the overall health of the T cells from Day 0. Thegenetically engineered T cells derived from these apheresed samples grewfaster, had higher transduction efficiencies, and maintained a morememory-like state when they are processed along with other cells in theapheresed sample, compared to the same apheresed cells that were furtherenriched for specific T cell populations.

It was found that further isolation, selection and/or enrichment for anysubset of the apheresed cells, such as by cell type or phenotype, wasnot necessary to achieve genetically engineered T cells with a highdegree of purity and viability, with high transgene expression, andstarting with this mixed cell population, did not impact the capabilityof generating cell densities well over 10 billion in a 10-day process.The resulting T cells were less differentiated and remained in a morememory-like phenotype which increases persistence and over all efficacy,as well as being more potent in killing target cells upon challenge.Starting with the apheresed cells eliminates additional separation stepssuch as magnetic separation or other sedimentation to isolate cells ofinterest, resulting in a continuous minimal manipulation operation fromapheresed donor cells to harvest of genetically engineered T cells. Thisdrastically reduces the per patient footprint inside a manufacturingfacility and the time for processing the harvested cells. In addition,eliminating the need for selection or enhancement of donor cells priorto activation, results in less manipulation of the donor sample,decreases exposure to unnecessary chemicals such as Ficoll®, reducesoverall cell loss, overall cost, and the possibility of damage to cells,and greatly diminished the risk of contaminating the patient sample.Autologous cell samples come from patients with grievous diseases. Thereis always a risk when taking a cell sample from these patients andadministering the resulting genetically engineered T cells back to thepatient. Exposing the patient to additional unnecessary risks byexcessive manipulation and exposure of the cells to unnecessaryprocessing, mishandling during the intensive hands on processing, oradministering contaminated cells back to the patient, is not a viableoption.

The invention provides a method for producing genetically engineeredautologous T cells expressing at least one protein of interest, themethod comprising inoculating a closed single use bioreactor bagcontaining culture media with apheresed donor cells and one or moresoluble T cell activators, wherein the bioreactor bag is part of arocking bioreactor platform, culturing the cells in the closed singleuse bioreactor bag continuously rocking at a rate of about 2 RPM,transducing the cells in the closed single use bioreactor bag with atleast one soluble viral vector comprising a polynucleotide which encodesthe protein of interest continuously rocking at a rate of about 2 RPM,and expanding the cells in the closed single use bioreactor bag at arocking rate of about 2 RPM and increasing the culture volume androcking the rate as needed to maintain the culture until harvest.

In one embodiment the apheresed donor cells comprise cells fromperipheral blood. In a related embodiment the apheresed donor cellscomprise nucleated and non-nucleated cells. In one embodiment theapheresed donor cells comprise leukocytes and erythrocytes. In a relatedembodiment the apheresed donor cells also comprise granulocytes and/orplatelets. In one embodiment the apheresis is leukapheresis. In arelated embodiment the apheresed donor cells are provided in aLeukopak®.

The apheresed donor cells are not subjected to any further isolation,selection and/or enrichment for any cell population(s) or cell type(s)following apheresis, and prior to activating the apheresed donor cells.

As will be appreciated, the harvested cells may be washed to remove theplasma fraction and any apheresis buffers, and to place the harvestedcells in an appropriate buffer or media for subsequent processing.Closed, automated processes are commercially available for this purposeand include Sepax C-Pro (GE Healthcare, Pittsburgh, Pa.), Cobe 2991 cellprocessor (Terumo BCT, Lakewood, Colo.), CellSaver 5 (Haemonetics,(Boston, Mass.)) and the like. In one embodiment the apheresed donorcells are washed and resuspended in a culture media prior toinoculation. No additional wash steps are performed until the expandedgenetically engineered autologous T cells are harvested for formulationand cryopreservation.

T cell activation is an antigen-dependent process resulting inproliferation and differentiation of naïve T cells into effector cells.Activation is stimulated by primary and coactivating signals. Withappropriate stimulation T cells will proliferate in vitro. T cellsrequire two signals to become fully activated, the primary stimulationis antigen specific, from interaction of the T cell receptor with apeptide-HLA molecule on an antigen presenting cell. The second isnon-antigen specific co-stimulatory signals from interaction between themembrane of the antigen presenting cell and the T cell.

As described herein, activation strength did not appear to correlatewith better transduction efficiency. Bound activators did not yield hightransduction rates despite providing the highest signal strength.Soluble activators, such as the mixtures of soluble CD3, CD28, and/orCD2 antibodies, particularly the combination of soluble CD3, CD28 andCD2 antibodies, had a signal strength falling between activators boundto bags and those bound to beads, however they activated T cells withthe highest expression of GFP and the engineered TCR.

One or more soluble T cell activators may be used to produce apopulation of activated T cells. Such T cell activators includeantibodies or functional fragment thereof that target T cell stimulatorand/or co-stimulatory molecules. Such T cell activators include, but arenot limited to, anti-CD3 antibodies or binding fragments, anti-CD28antibodies or binding fragments, and anti-CD2 antibodies or bindingfragments, or combinations thereof. Also included are anti-human CD3monospecific tetrameric antibody complex, an anti-human CD28monospecific tetrameric antibody complex, and an anti-human CD2monospecific tetrameric antibody complex. Such T cell activators arecommercially available from a variety of sources including Stem CellTechnologies, Vancouver, BC CA, among others.

In one embodiment at least one T cell activator is an anti CD3 antibodyor binding fragments thereof. In one embodiment the T cell activatorcomprises an anti CD3 antibody and an anti CD28 antibody, or bindingfragments thereof. In another embodiment the T cell activator comprisesat least an anti CD3 antibody, an anti CD28 antibody, and an anti CD2antibody, or binding fragments thereof. In another embodiment the T cellactivator comprises at least an anti-human CD3 monospecific tetramericantibody complex, an anti-human CD28 monospecific tetrameric antibodycomplex, and an anti-human CD2 monospecific tetrameric antibody complex.

In one embodiment the concentration of at least one soluble T cellactivator is at least 0.001 μg/ml to at least 10 μg/ml. In a relatedembodiment the concentration is at least 0.01 μg/ml to at least 10μg/ml. In a related embodiment the concentration is at least 0.1 μg/mlto at least 10 μg/ml. In a related embodiment the concentration is atleast 1 μg/ml to at least 10 μg/ml. In a related embodiment theconcentration is at least 5 μg/ml to at least 10 μg/ml. In oneembodiment the concentration of at least one T cell activator is atleast 0.001 μg/ml to at least 1 μg/ml. In a related embodiment theconcentration of at least one T cell activator is at least 0.01 μg/ml toat least 1 μg/ml. In a related embodiment the concentration of at leastone T cell activator is at least 0.1 μg/ml to at least 1 μg/ml. In oneembodiment the concentration of at least one T cell activator is atleast 0.001 μg/ml to at least 5 μg/ml. In a related embodiment theconcentration of at least one T cell activator is at least 0.01 μg/ml toat least 5 μg/ml. In a related embodiment the concentration of at leastone T cell activator is at least 0.1 μg/ml to at least 5 μg/ml. In arelated embodiment the concentration of at least on T cell activator isat least 1 μg/ml to at least 5 μg/ml. In one embodiment theconcentration is at least 0.001 μg/ml. In one embodiment theconcentration is at least 0.01 μg/ml. In one embodiment theconcentration is at least 0.1 μg/ml. In one embodiment the concentrationis at least 1 μg/ml. In one embodiment the concentration is at least 5μg/ml. In one embodiment the concentration is at least 10 μg/ml. In oneembodiment the concentration greater than 10 μg/ml.

In one embodiment the wherein the culture media comprises at least onesoluble cytokine. Cytokines, such as IL-1, IL-2, IL-4, IL-5, IL-7,IL-15, and/or IL-21, may also be used as soluble T cell stimulatingagents.

In one embodiment the soluble cytokine selected from IL-2, IL-7, IL-15,or IL-21. In one embodiment the soluble cytokine is IL-7 in combinationwith IL-15 or IL-21. Such cytokines may be used at concentrations fromat least 5 ng/ml to at least 30 ng/ml or more. In one embodiment, theconcentration of at least one cytokine is at least 5 ng/ml to at least25 ng/ml. In one embodiment the concentration of at least one cytokineis at least 5 ng/ml to at least 20 ng·ml. In one embodiment theconcentration of at least one cytokine is at least 5 ng/ml to at least15 ng/ml. In one embodiment the concentration of at least one cytokineis at least 5 ng/ml to at least 10 ng/ml. In one embodiment theconcentration of at least one cytokine is at least 10 ng/ml to at least20 ng/ml. In one embodiment the concentration of at least one cytokineis at least 5 ng/ml. In one embodiment the concentration of at least onecytokine is at least 10 ng/ml. In one embodiment the concentration of atleast one cytokine is at least 15 ng/ml. In one embodiment theconcentration of at least one cytokine is at least 20 ng/ml. In oneembodiment the concentration of at least one cytokine is at least 25ng/ml. In one embodiment the concentration of at least one cytokine isat least 30 ng/ml. In one embodiment the concentration of at least onecytokine is greater than 30 ng/ml. Other molecules that impact T cellactivation or maturation may also be included, such as a WNT pathwayactivator. Such activators include 4,6-disubstituted pyrrolopyrimidineTWS119, an inhibitor of the serine/threonine kinaseglycogen-synthase-kinase-3β(Gsk-3β), (Stemcell Technologies). In oneembodiment the concentration of TWS119 is at least 5 μM to at least 20μM or more. In one embodiment the concentration of TWS119 is at least 5μM to at least 15 μM. In one embodiment the concentration of TWS119 isat least 5 μM to at least 10 μM. In one embodiment the concentration ofTWS119 is at least 10 μM to at least 20 μM. In one embodiment theconcentration of TWS119 is at least 5 μM. In one embodiment theconcentration of TWS119 is at least 10 μM. In one embodiment theconcentration of TWS119 is at least 15 μM. In one embodiment theconcentration of TWS119 is at least 20 μM. In one embodiment theconcentration of TWS119 is greater than 20 μM. In one embodiment theculture media comprises a mixture of soluble TWS117, IL-7, and IL-21.

In one embodiment least one soluble cytokine is IL-2. In one embodimentthe IL-2 is at a concentration of about 250 IU/ml to about 350 IU/ml. Inone embodiment the soluble cytokine is IL-2 at a concentration of about250 IU/ml to 300 IU/ml. In one embodiment the soluble cytokine is IL-2at a concentration of about 300 IU/ml to 350 IU/ml. In one embodimentthe soluble cytokine is IL-2 at a concentration of about 250 IU/ml, 300IU/ml, or 350 IU/ml. In one embodiment the soluble cytokine is IL-2 at aconcentration of about 250 IU/ml. In one embodiment the soluble cytokineis IL-2 at a concentration of about 300 IU/ml. In one embodiment thesoluble cytokine is IL-2 at a concentration of about 350 IU/ml.

During antigen stimulation, T cells shift to a glycolytic metabolism tosustain effector function. This happens in the first few days afteradding activators to the cells. Glycolysis inhibitors can be added withthe activators to inhibit glycolysis metabolism during the activationand transduction steps, which can contribute to generation of T cellswith less differentiated phenotypes, enhanced transduction efficiency,and/or enhanced T cell expression. As described herein, the engineered Tcells derived from 2-deoxy-D-glucose (2-DG)-inhibited T cells producedmore Tscm and Tcm compared with the T cells derived from the mediawithout 2-DG. The inhibitor can be kept in the culture media untilharvest or removed at any point to restore glycolysis and support T cellexpansion. In one embodiment the culture media also comprises a solubleglycolysis inhibitor. In a related embodiment the soluble glycolysisinhibitor is 2-deoxy-D-glucose (2-DG). In one embodiment theconcentration of 2-DG is about 1 mM to about 3 mM. In a relatedembodiment the concentration of 2-DG is about 1 mM to about 2 mM In arelated embodiment the concentration of 2-DG is about 2 mM to about 3mM. In one embodiment the concentration of 2-DG is about 1 mM or less.In one embodiment the concentration is about 2 mM. In one embodiment theconcentration of 2-DG is about 3 mM or more.

Procedures for producing engineered T cells comprise many unitoperations such as cell isolation, selection and/or enrichment of theharvested donor sample for a particular cell type or phenotype,activation, transduction, expansion, harvest, formulation, andcryopreservation. During each of these steps, multiple vessels orprocesses may be used to perform each operation. For example, to obtaina desired cell type or phenotype, one or more closed vessels and/orprocesses may be used to perform steps such as washes, magnetic-antibodylabeling, performing selection or enrichments procedures,centrifugation/sedimentation, and concentration of the isolated,selected, or enriched cells. Much use is made of bound activators foractivation and/or bound agents to enhance transduction, whether they arecoated on plates, or bags or bound to beads or other supports. Once thestimulation or transduction using these bound activators is complete,the cells must be removed, washed, and transferred to new vessels.Multiple vessels may be used during expansion to accommodate increaseddemand for nutrients and increasing culture volume. Switches betweendifferent types of vessels associated with these steps is time-consumingand costly and may expose the cells to damage, loss and contamination.

As described herein, the invention provides a closed continuousoperation from inoculation of apheresed donor cells all the way throughto the harvest of expanded genetically engineered T cells. All steps,including activation, transduction, and expansion, take place in asingle closed bioreactor system that is constantly rocked at a speed ofat least 2 RPM. This not only minimizes manual manipulation of donormaterial, reducing risk of contamination and cell loss, it allows forautomated feeding and culture manipulation. Additional equipment, suchas incubators and separate vessels for activation and/or transduction,are not necessary to process the donor's cells which drastically reducesthe risk of contamination and cells loss, but also reduced the cost inmaterials/reagents and FTE time, and the per patient footprint inside amanufacturing facility.

As described herein, activation, transduction, and expansion in acontinuously rocking bioreactor that makes use of soluble components ina culture media optimized for T cell growth, resulted in a high-densityproduction of engineered autologous T cells from washed apheresed donorcells. Use of soluble components such as T cell activators, stimulators,and metabolic pathway inhibitors, allowed for a continuous flow betweenthe various steps in the manufacture of the autologous engineered Tcells and produced engineered T cells at a desired phenotype andtransgene expression. Rocking during this process also ensured asufficient mass transfer of oxygen and nutrients to support high-densitycultures in contrast to static cultures which must be grown at low celldensity (≤2E6 cells/ml) due to limitation of the culture media understatic conditions. While the rocking bioreactors and commonly usedG-Rex® gas permeable culture systems both have efficient oxygentransport, the G-Rex® systems lacks the convection flow that transfersnutrients to cells in a timely manner. Also, using the rockingbioreactor system allows for perfusion of fresh media which enable veryhigh cell densities, up to 50E6 cells/ml in some cases.

Activation, transduction, and expansion of autologous donor apheresedcells comprising leukocytes and erythrocytes, was carried out in acontinuous closed system within a single bioreactor bag that was rockedcontinuously. Bioreactor bags, such as Cellbag bioreactor bags (GEHealthcare) are used as part of rocking bioreactor platforms such asWave or Xuri Cell Expansion systems (GE Healthcare), and in addition tooperating as a bioreactor also have the capability of rocking at variousspeeds and angles which allows efficient mass transfer of oxygen andnutrients without introducing too much shear stress to T cells. Suchbioreactor bags are typically equipped with perfusion filters to filterincoming culture media, ports and lines for adding culture media andother components, withdrawing spent media and samples, all within asterile controlled environment. The bags are also equipped to connectwith controls on the rocking platform system that automatically measureand control culture parameters such as temperature, CO₂, O₂, pH andmetabolic and media parameters such as glucose and lactate, as well ascontrolling gas and media flow. The bags allow for automated feeding, beit bolus, fed batch, fed batch/perfusion, and/or semi- or continuousperfusion. In addition to reducing the amount of manual manipulation ofcells, culturing the cells in a closed bioreactor system allows forgreater capacity and improvement to the yield and culture density of theengineered T cells.

Production of engineered autologous T cells for cell therapy indicationstypically relies on static cultures that make use of gas permeablevessels can require extensive manual manipulation, exposing the culturesand the equipment to contamination risk as well as limiting nutrientflow to cells. Static, gas permeable vessels, such as bags, flasks, andplates, are widely used in autologous T cell processes that involve atransduction step to produce genetically engineered T cells. Examples ofsuch static gas permeable cell culture bags currently used includePermalife Cell Culture Bags, Origin Biomedical, Austin Tex., MACS GMPCell Differentiation Bags, Miltenyi Biotech, Cambridge, Mass.), RapidExpansion Flask (G-Rex) bioreactor, other G-Rex® vessels Wilson-WolfManufacturing. The vessels require an incubator or otherclimate-controlled chamber to maintain desired temperatures and are gaspermeable to allow passive diffusion of O₂ from the ambient incubatorenvironment and CO₂ from the media. The vessels require manualmanipulation to move them to and from processing equipment, incubators,or biosafety cabinets, and the like that are necessary for maintainingthe cultures. In addition, the vessels require constant connecting andreconnecting to processing equipment, syringes, pumps, chambers, andother vessels and devices to maintain the cultures. The manualprocessing increases the risk of contamination to the cultures. Inaddition, Permalife Cell Culture bags are only designed for low densityT cell expansion (<2E6 cells/ml) and will require extremely largevolumes to supply at the high-dosages required for T cell therapy.G-Rex®, on the other hand, could expand T cells in a relatively highdensity (˜10E6 cells/ml) but are challenged with any manipulation ofexpansion parameters. These two vessels are also not equipped withcontrol systems and data recording ability to support a more advancedand digital manufacturing operation.

The inventive method provides inoculating a closed single use bioreactorbag containing culture media with apheresed donor cells and one or moresoluble T cell activators, wherein the bioreactor bag is part of arocking bioreactor platform. The volume of culture media in thebioreactor bag at the time of inoculation can be any predeterminedvolume. In one embodiment the volume of culture media is at least theminimal volume of the bioreactor bag. In one embodiment the secondvolume of culture media is at least 300 ml. In one embodiment the volumeof the culture media 400 ml or more. In one embodiment the volume ofculture media is about 300 ml to about 400 ml. In one embodiment thevolume of the culture media is about 325 ml to about 400 ml. In oneembodiment the volume of the culture media is about 350 ml to about 400ml. In one embodiment the volume of the culture media is about 375 ml toabout 400 ml. In one embodiment the volume of culture media is about 300ml, about 325 ml, about 350 ml, about 375 ml, or about 400 ml. In oneembodiment the volume of culture media is about 300 ml. In oneembodiment the volume of culture media is about 325 ml. In oneembodiment the volume of culture media is about 350 ml. In oneembodiment the volume of culture media about 375 ml. In one embodimentthe volume of culture media is about 400 ml. In one embodiment thevolume of culture media is about 400 ml or more.

T cells require cell-cell contact to activate and survive. Aninsufficient concentration of cells during activation could reducecell-cell contact to a level associated with poor activation. Theapheresed donor cells comprise nucleated and non-nucleated cells. Thenumber of nucleated cells in the apheresed donor sample can bedetermined by any known method, including the use of automated systemssuch as a NC-200™ Automated Cell Counter (ChemMetec, Denmark). In oneembodiment the apheresed donor cells comprise a sufficient number ofnucleated cells for activation in the bioreactor bag at a rocking speedof 2 RPM. In one embodiment the apheresed donor cells comprise about1.0E9 to 1.3E9 nucleated cells. In a related embodiment the aphereseddonor cells comprise about 1.1E9 to about 1.2E9 nucleated cells. In arelated embodiment the apheresed donor cells comprise about 1.2E9 toabout 1.3E9 nucleated cells. In a related embodiment the apheresed donorcells comprise about 1.2E9 to about 1.25E9 nucleated cells. In a relatedembodiment the apheresed donor cells comprise about 1.0E9 nucleatedcells. In a related embodiment the apheresed donor cells comprise about1.1E9 nucleated cells. In a related embodiment the apheresed donor cellscomprise about 1.2E9 nucleated cells. In a related embodiment theapheresed donor cells comprise about 1.25E9 nucleated cells. In arelated embodiment the apheresed donor cells comprise about 1.3E9nucleated cells. In a related embodiment the apheresed donor cellscomprise about 1.0E6 nucleated cells, about 1.1E9 nucleated cells, about1.2E9 nucleated cells, about 1.25E9 nucleated cells, or about 1.3E9nucleated cells.

In one embodiment the invention provides inoculating a closed single usebioreactor bag containing culture media with apheresed donor cells at acell density of about 1E6 to about 5E6 nucleated cells/ml. In a relatedembodiment the cell density of about 2E6 to about 4E6 nucleatedcells/ml. In one embodiment the cell density is about 3E6 to about 4E6nucleated cells/ml. In one embodiment the cell density is about 3E6 toabout 3.75E6 nucleated cells/ml. In one embodiment the cell density isabout 3E6 to about 3.5E6 nucleated cells/ml. In one embodiment the celldensity is about 3E6 to about 3.35E6 nucleated cells/ml. In oneembodiment the cell density is about 2E6 to about 3E6 nucleatedcells/ml. In one embodiment the cell density is about 2E6 nucleatedcells/ml, about 3E6 nucleated cells/ml, about 3.25E6 nucleated cells/ml,about 3.75E6 nucleated cells/ml, or about 4E6 nucleated cells/ml. In oneembodiment the cell density is about 2E6 nucleated cells/ml. In oneembodiment the cell density is about 3E6 nucleated cells/ml. In oneembodiment the cell density is about 3.25E6 nucleated cells/ml. In oneembodiment the cell density is about 3.75E6 nucleated cells/ml. In oneembodiment the cell density is about 4E6 nucleated cells/ml. In oneembodiment the cell density is about 3E6 nucleated cells/ml to 4E6nucleated cells/ml in about 300 ml culture media containing a solublecytokine.

The during activation cells can be cultured at a predeterminedtemperature. In one embodiment, the cells are cultured at 34-39° C. Inone embodiment, the cells are cultured at 34-35° C. In one embodiment,the cells are cultured at 35-37° C. In one embodiment, the cells arecultured at 35-36° C. In one embodiment, the cells are cultured at36-37° C. In one embodiment, the cells are cultured at 34° C., 35° C.,36° C., or 37° C. In one embodiment, the cells are cultured at 34° C. Inone embodiment, the cells are cultured at 35° C. In one embodiment, thecells are cultured at 36° C. In one embodiment, the cells are culturedat performed at 37° C.

The during activation cells can be cultured for a predetermined time. Inone embodiment, the cells are cultured for at least 12 hours. In oneembodiment, the cells are cultured for at least 24 hours. In oneembodiment the cells are cultured for at least 12 to at least 24 hours.In one embodiment the cells are cultured for at least 12 to at least 20hours. In one embodiment the cells are cultured for at least 12 to atleast 18 hours. In one embodiment the cells are cultured for at least 12to at least 16 hours. In one embodiment the cells are cultured for atleast 12 to at least 14 hours. In one embodiment the cells are culturedfor at least 14 to at least 24 hours. In one embodiment the cells arecultured for at least 14 to at least 20 hours. On one embodiment thecells are cultured for at least 14 to at least 18 hours. On oneembodiment the cells are cultured for at least 14 to at least 16 hours.On one embodiment the cells are cultured for at least 16 to at least 24hours. On one embodiment the cells are cultured for at least 16 to atleast 20 hours. In one embodiment the cells are cultured for at least 16to at least 18 hours. On one embodiment the cells are cultured for atleast 18 to at least 24 hours. In one embodiment the cells are culturedfor at least 18 to 20 hours. In one embodiment the cells are culturedfor at least 20 to at least 24 hours. In one embodiment the cells arecultured for at least 12 hours. In one embodiment the cells are culturedfor at least 13 hours. In one embodiment the cells are cultured for atleast 14 hours. In one embodiment the cells are cultured for at least 15hours. In one embodiment the cells are cultured for at least 16 hours.In one embodiment the cells are cultured for at least 17 hours. In oneembodiment the cells are cultured for at least 18 hours. In oneembodiment the cells are cultured for at least 19 hours. In oneembodiment the cells are cultured for at least 20 hours. In oneembodiment the cells are cultured for at least 21 hours. In oneembodiment the cells are cultured for at least 22 hours. In oneembodiment the cells are cultured for at least 23 hours. In oneembodiment the cells are cultured for at least 24 hours.

In one embodiment of the invention, at least one soluble T-cellactivator is bound to at least one donor cell at the time ofinoculation. In one embodiment the apheresed donor cells are incubatedwith one or more soluble T cell activators prior to inoculating into thebioreactor bag. In one embodiment the incubation is for a sufficienttime to allow for saturation of binding of one or more soluble T cellactivators to the apheresed donor cells prior to inoculation. In oneembodiment the apheresed donor cells are incubated with one or more Tcell activators for at least 30 minutes or more. In one embodiment theapheresed donor cells are incubated with one or more T cell activatorsfor at least 30 minutes to at least 2 hours or more. In a relatedembodiment the apheresed donor cells are incubated with one or more Tcell activators for at least 1 hour to at least 2 hours. In oneembodiment the incubation is at least 30 minutes. In one embodiment theincubation is about 1 hour. In one embodiment the incubation is about 2hours.

In one embodiment the apheresed donor cells and one or more soluble Tcell activators are incubated in a transfer bag. A minimal amount ofculture media, enough to suspend the cells and the soluble T cellactivator and allow for binding of the activator to the nucleated cells,is used, preferably enough to keep the total volume of cells. In oneembodiment the volume of culture media in the transfer bag is about 5 mlto about 50 ml. In one embodiment of the invention, the initial volumeof culture media comprising a soluble cytokine and one or more T cellactivators is about 5 ml to about 40 ml. In one embodiment of theinvention, the initial volume of culture media comprising a solublecytokine and one or more T cell activators is about 5 ml to about 30 ml.In one embodiment of the invention, the initial volume of culture mediacomprising a soluble cytokine and one or more T cell activators is about5 ml to about 20 ml. In one embodiment of the invention, the initialvolume of culture media comprising a soluble cytokine and one or more Tcell activators is about 5 ml to about 15 ml. In one embodiment of theinvention, the initial volume of culture media comprising a solublecytokine and one or more T cell activators is about 5 ml to about 14 ml.In one embodiment of the invention, the initial volume of culture mediacomprising a soluble cytokine and one or more T cell activators is about5 ml to about 13 ml. In one embodiment of the invention, the initialvolume of culture media comprising a soluble cytokine and one or more Tcell activators is about 5 ml to about 12 ml. In one embodiment of theinvention, the initial volume of culture media comprising a solublecytokine and one or more T cell activators is about 5 ml to about 11 ml.In one embodiment of the invention, the initial volume of culture mediacomprising a soluble cytokine and one or more T cell activators is about5 ml to about 10 ml. In one embodiment of the invention, the initialvolume of culture media comprising a soluble cytokine and one or more Tcell activators is about 5 ml to about 9 ml. In one embodiment of theinvention, the initial volume of culture media comprising a solublecytokine and one or more T cell activators is about 5 ml to about 8 ml.In one embodiment of the invention, the initial volume of culture mediacomprising a soluble cytokine and one or more T cell activators is about5 ml to about 7 ml. In one embodiment of the invention, the initialvolume of culture media comprising a soluble cytokine and one or more Tcell activators is about 5 ml to about 6 ml. In one embodiment of theinvention, the initial volume of culture media comprising a solublecytokine and one or more T cell activators is about 8 ml, about 6 ml,about 7 ml, about 8 ml, about 9 ml, about 10 ml, about 15 ml, about 20ml, about 30 ml, about 40 ml, or about 50 ml.

Transduction refers to the process whereby foreign DNA is introducedinto a cell via viral vector. See Jones et al., (1998). Genetics:principles and analysis. Boston: Jones & Bartlett Publ. As used herein,“vector” means any molecule or entity such as a viral a vector, that isuseful for transformation of a host cell, such as a T cell, and containnucleic acid sequences that direct and/or control (in conjunction withthe cell) expression of one or more heterologous coding regionsoperatively linked thereto. An expression construct may include, but isnot limited to, sequences that affect or control transcription,translation, and, if introns are present, affect RNA splicing of acoding region operably linked thereto. One or more vectors are theninserted into the cell for amplification and/or polypeptide expression.The transformation of an expression vector into a selected cell may beaccomplished through viral transformation. Viral transformed T cells canbe obtained by transducing cells with a viral vector comprising apolynucleotide which encodes the protein of interest. Viral vectorsinclude retroviral vectors, murine leukemia virus vectors, SFG vectors,adenoviral vectors, lentiviral vectors, adeno-associated virus (AAV)vectors, Herpes virus vectors, and vaccinia virus vectors.

The invention provides transducing the cells in the closed single usebioreactor bag with at least one viral vector comprising apolynucleotide which encodes the protein of interest while continuouslyrocking at a rate of 2 RPM. The continuous rocking rate of 2 RPM allowedfor sufficient cell-cell contact, necessary for successfultransformation. In one embodiment the culture is rocked at a rate of 2RMP at a 2° angle.

In one embodiment, the viral vector is a retroviral vector. Retroviralvectors, such as lentivirus vectors, persist in the nucleus asintegrated provirus and reproduce with cell division. In one embodimentthe viral vector is a lentiviral vector.

In one embodiment the cells can be engineered using one or more viralvectors comprising one or more polynucleotide sequences encoding one ormore proteins of interest. In one embodiment the protein of interest iscell surface receptor. In one embodiment the cell surface receptor is achimeric antigen receptor or a T cell receptor. In one embodiment thecell surface receptor recognizes an antigenic target on the surface of atarget cell.

In one embodiment the lentiviral vector is added at a multiplicity ofinfection (MOI) of 0.25-10. In one embodiment the lentiviral vector isadded at a MOI of 0.25-5. In one embodiment the lentiviral vector isadded at a MOI of 0.25-2. In one embodiment the lentiviral vector isadded at a MOI of 0.25-1. In one embodiment the lentiviral vector isadded at a MOI of 0.25-0.5. In one embodiment the lentiviral vector isadded at a MOI of 0.5-10. In one embodiment the lentiviral vector isadded at a MOI of 0.5-5. In one embodiment the lentiviral vector isadded at a MOI of 0.5-2. In one embodiment the lentiviral vector isadded at a MOI of 0.5-1. In one embodiment the lentiviral vector isadded at a MOI of 1-10. In one embodiment the lentiviral vector is addedat a MOI of 1-5. In one embodiment the lentiviral vector is added at aMOI of 1-2. In one embodiment the lentiviral vector is added at a MOI of2-10. In one embodiment the lentiviral vector is added at a MOI of 2-5.In one embodiment the lentiviral vector is added at a MOI of 5-10. Inone embodiment the lentiviral vector is added at a MOI of 0.25, 0.5, 1,2, 5, or 10. In one embodiment the lentiviral vector is added at a MOIof 10. In one embodiment the lentiviral vector is added at a MOI of 5.In one embodiment the lentiviral vector is added at a MOI of 2. In oneembodiment the lentiviral vector is added at a MOI of 1. In oneembodiment the lentiviral vector is added at a MOI of 0.5. In oneembodiment the lentiviral vector is added at a MOI of 0.25.

The cells can be transduced at a predetermined temperature. In oneembodiment, the cells are transduced at 34-39° C. In one embodiment, thecells are transduced at 34-35° C. In one embodiment, the cells aretransduced at 35-37° C. In one embodiment, the cells are transduced at35-36° C. In one embodiment, the cells are transduced at 36-37° C. Inone embodiment, the cells are transduced at 34° C., 35° C., 36° C., or37° C. In one embodiment, the cells are transduced at 34° C. In oneembodiment, the cells are transduced at 35° C. In one embodiment, thecells are transduced at 36° C. In one embodiment, the cells aretransduced at performed at 37° C.

The cells can be transduced for a predetermined time. In one embodiment,the cells are transduced for at least 12 hours. In one embodiment, thecells are transduced for at least 24 hours. In one embodiment the cellsare transduced for at least 12 to at least 24 hours. In one embodimentthe cells are transduced for at least 12 to at least 20 hours. In oneembodiment the cells are transduced for at least 12 to at least 18hours. In one embodiment the cells are transduced for at least 12 to atleast 16 hours. In one embodiment the cells are transduced for at least12 to at least 14 hours. In one embodiment the cells are transduced forat least 14 to at least 24 hours. In one embodiment the cells aretransduced for at least 14 to at least 20 hours. On one embodiment thecells are transduced for at least 14 to at least 18 hours. On oneembodiment the cells are transduced for at least 14 to at least 16hours. On one embodiment the cells are transduced for at least 16 to atleast 24 hours. On one embodiment the cells are transduced for at least16 to at least 20 hours. In one embodiment the cells are transduced forat least 16 to at least 18 hours. On one embodiment the cells aretransduced for at least 18 to at least 24 hours. In one embodiment thecells are transduced for at least 18 to 20 hours. In one embodiment thecells are transduced for at least 20 to at least 24 hours. In oneembodiment the cells are transduced for at least 12 hours. In oneembodiment the cells are transduced for at least 13 hours. In oneembodiment the cells are transduced for at least 14 hours. In oneembodiment the cells are transduced for at least 15 hours. In oneembodiment the cells are transduced for at least 16 hours. In oneembodiment the cells are transduced for at least 17 hours. In oneembodiment the cells are transduced for at least 18 hours. In oneembodiment the cells are transduced for at least 19 hours. In oneembodiment the cells are transduced for at least 20 hours. In oneembodiment the cells are transduced for at least 21 hours. In oneembodiment the cells are transduced for at least 22 hours. In oneembodiment the cells are transduced for at least 23 hours. In oneembodiment the cells are transduced for at least 24 hours.

As described herein, transduction was performed using a rockingbioreactor equipped with a single use bioreactor bag at a rocking rateof 2 RPM, in particular, a rocking rate of 2 RPM at an angle of 2°. Oneor more viral vectors comprising polynucleotides encoding proteins ofinterest may be directly inoculated into the culture in the bioreactorbag while the bag is rocking. Use of bags impermeable to gas such asthose as used on rocking bioreactors has not been recommended for useduring transduction. “Vessels that are impermeable to gas, such asdisposable bioreactors that are designed to fit rocking motionbioreactors, are not ideal for transduction of cells via viral vectors.Therefore, transduction is currently carried out in static cell culturebags or planar vessels.”, see Farid and Jenkins, Bioprocesses for CellTherapies, Chapter 44, Biopharmeceutical Processing: Development, Designand Implementation of Manufacturing Processes, Eds. Jagschies et al.,Elsevier, page 914, 2018.

However, as described herein, compared to cells transduced in static gaspermeable vessels, the cells transduced in disposable bioreactors bagsdesigned to be part of rocking motion bioreactors, had a greatertransduction efficiency, were less differentiated, more memory-celllike, and had greater potency in killing target cells than those cellstransduced in a static gas permeable bag or in a static G-Rex® gaspermeable system.

In one embodiment, following transduction, half of the culture media isremoved from the bioreactor bag and replaced with an equal volume offresh culture media and rocked at a rate of 2 RPM. In one embodiment theculture is rocked at a rate of 2 RMP at a 2° angle.

The cells can be cultured at a predetermined temperature. In oneembodiment, the cells are cultured at 34-39° C. In one embodiment, thecells are cultured at 34-35° C. In one embodiment, the cells arecultured at 35-37° C. In one embodiment, the cells are cultured at35-36° C. In one embodiment, the cells are cultured at 36-37° C. In oneembodiment, the cells are cultured at 34° C., 35° C., 36° C., or 37° C.In one embodiment, the cells are cultured at 34° C. In one embodiment,the cells are cultured at 35° C. In one embodiment, the cells arecultured at 36° C. In one embodiment, the cells are cultured atperformed at 37° C.

The cells can be cultured for a predetermined time. In one embodiment,the cells are cultured for at least 12 hours. In one embodiment, thecells are cultured for at least 24 hours. In one embodiment the cellsare cultured for at least 12 to at least 24 hours. In one embodiment thecells are cultured for at least 12 to at least 20 hours. In oneembodiment the cells are cultured for at least 12 to at least 18 hours.In one embodiment the cells are cultured for at least 12 to at least 16hours. In one embodiment the cells are cultured for at least 12 to atleast 14 hours. In one embodiment the cells are cultured for at least 14to at least 24 hours. In one embodiment the cells are cultured for atleast 14 to at least 20 hours. On one embodiment the cells are culturedfor at least 14 to at least 18 hours. On one embodiment the cells arecultured for at least 14 to at least 16 hours. On one embodiment thecells are cultured for at least 16 to at least 24 hours. On oneembodiment the cells are cultured for at least 16 to at least 20 hours.In one embodiment the cells are cultured for at least 16 to at least 18hours. On one embodiment the cells are cultured for at least 18 to atleast 24 hours. In one embodiment the cells are cultured for at least 18to 20 hours. In one embodiment the cells are cultured for at least 20 toat least 24 hours. In one embodiment the cells are cultured for at least12 hours. In one embodiment the cells are cultured for at least 13hours. In one embodiment the cells are cultured for at least 14 hours.In one embodiment the cells are cultured for at least 15 hours. In oneembodiment the cells are cultured for at least 16 hours. In oneembodiment the cells are cultured for at least 17 hours. In oneembodiment the cells are cultured for at least 18 hours. In oneembodiment the cells are cultured for at least 19 hours. In oneembodiment the cells are cultured for at least 20 hours. In oneembodiment the cells are cultured for at least 21 hours. In oneembodiment the cells are cultured for at least 22 hours. In oneembodiment the cells are cultured for at least 23 hours. In oneembodiment the cells are cultured for at least 24 hours.

The invention provides expanding the cells in the closed single usebioreactor bag at a rocking rate of about 2 RPM and increasing theculture volume and rocking the rate as needed to maintain the cultureuntil harvest. T cells require cell-cell contact to survive. Less than2E6 cells/ml can deprive cell-cell contact, increase cell stress, and/ordecrease ability to overcome bioreactor agitation and to proliferate.Scale-up to 4E6 cells/ml or more allows for timely increases in theculture volume, which also simplifies the feeding process. Stepwiseincreased in culture volume allow for scale up of the culture to 1 literin a timely manner, with rocking speed/angle corresponding to increasesin volume to allow for a steady amount of dissolve oxygen in the systemto facilitate cell expansion. During activation, T cells consume andrelease large amounts of glucose and lactate. High levels of lactateappear to be detrimental for T cell activation and expansion. As aresult, cell media exchange is not a parameter corresponding to celldensity in the early phase of activation and transduction as the cellsare not expanding much. During activation/transduction, lactate level isthe critical culture parameter and sufficient turnover of media is acritical for T cell activation and expansion. Semi-continuous mediaexchange is used to control metabolite level in the culture media.

T cells can reach a higher density when cultured in a rocking bioreactorplatform than in static gas permeable vessel system. Final yield for theprocess described herein, was between 15 million to 35 billion cells ina 1 L bioreactor in 10-14 days. One bioreactor is sufficient to supportone patient. To achieve the same 10 billion engineered T cells asproduced in a single liter produced by the inventive method wouldrequire ˜20 permeable bags (250 ml) or a 4 L G®-rex bioreactor. The cellyield of the inventive method is higher than that of other commonprocess in field.

In one embodiment, as the cells expand the volume of the culture mediais incrementally increased to maintain a cell density of at least 2E6nucleated cells/ml. In one embodiment the cells expand the volume of theculture media in bioreactor is incrementally increased to 1 liter duringexpansion to maintain a cell density of at least 4E6 nucleated cells/ml.In one embodiment the cell density during expansion is at least 2E6cells/ml or higher. In one embodiment the cell density is about 2E6cells/ml to about 3.75E6 cells/ml. In one embodiment the cell density isabout 2E6 cells/ml to about 3.50E6 cells/ml. In one embodiment the celldensity is about 2E6 cells/ml to about 3.25E6 cells/ml. In oneembodiment the cell density is about 2E6 cells/ml to about 3.00E6cells/ml. In one embodiment the cell density is about 2E6 cells/ml toabout 2.75E6 cells/ml. In one embodiment the cell density is about 2E6cells/ml to about 2.50E6 cells/ml. In one embodiment the cell density isabout 2E6 cells/ml to about 2.25E6 cells/ml. In one embodiment the celldensity is about 2.25E6 cells/ml to about 4.0E6 cells/ml. In oneembodiment the cell density is about 2.25E6 cells/ml to about 3.75E6cells/ml. In one embodiment the cell density is about 2.25E6 cells/ml toabout 3.50E6 cells/ml. In one embodiment the cell density is about2.25E6 cells/ml to about 3.25E6 cells/ml. In one embodiment the celldensity is about 2.25E6 cells/ml to about 3.0E6 cells/ml. In oneembodiment the cell density is about 2.25E6 cells/ml to about 2.75E6cells/ml. In one embodiment the cell density is about 2.25E6 cells/ml toabout 2.50E6 cells/ml. In one embodiment the cell density is about2.50E6 cells/ml to about 4.0E6 cells/ml. In one embodiment the celldensity is about 2.50E6 cells/ml to about 3.75E6 cells/ml. In oneembodiment the cell density is about 2.50E6 cells/ml to about 3.50E6cells/ml. In one embodiment the cell density is about 2.50E6 cells/ml toabout 3.25E6 cells/ml. In one embodiment the cell density is about2.50E6 cells/ml to about 3.00E6 cells/ml. In one embodiment the celldensity is about 2.50E6 cells/ml to about 2.75E6 cells/ml. In oneembodiment the cell density is about 2.50E6 cells/ml to about 2.50E6cells/ml. In one embodiment the cell density is about 2.50E6 cells/ml toabout 2.75E6 cells/ml. In one embodiment the cell density is about2.75E6 cells/ml to about 4.0E6 cells/ml. In one embodiment the celldensity is about 2.75E6 cells/ml to about 3.75E6 cells/ml. In oneembodiment the cell density is about 2.75E6 cells/ml to about 3.50E6cells/ml. In one embodiment the cell density is about 2.75E6 cells/ml toabout 3.25E6 cells/ml. In one embodiment the cell density is about2.75E6 cells/ml to about 3.00E6 cells/ml. In one embodiment the celldensity is about 2.75E6 cells/ml to about 2.75E6 cells/ml. In oneembodiment the cell density is about 2.75E6 cells/ml to about 2.50E6cells/ml. In one embodiment the cell density is about 3.0E6 cells/ml toabout 4.0E6 cells/ml. In one embodiment the cell density is about 3.0E6cells/ml to about 3.75E6 cells/ml. In one embodiment the cell density isabout 3.0E6 cells/ml to about 3.50E6 cells/ml. In one embodiment thecell density is about 3.0E6 cells/ml to about 3.25E6 cells/ml. In oneembodiment the cell density is about 3.25E6 cells/ml to about 4.0E6cells/ml. In one embodiment the cell density is about 3.25E6 cells/ml toabout 3.75E6 cells/ml. In one embodiment the cell density is about3.25E6 cells/ml to about 4.0E6 cells/ml. In one embodiment the celldensity is about 3.50E6 cells/ml to about 4.0E6 cells/ml. In oneembodiment the cell density is about 3.50E6 cells/ml to about 3.75E6cells/ml. In one embodiment the cell density is about 3.75E6 cells/ml toabout 4.0E6 cells/ml.

In one embodiment, during expansion, fresh culture media is added to thebioreactor by a combination of fed batch/perfusion feeding and/or byperfusion. In one embodiment, fresh culture media is added by fedbatch/perfusion until the culture reaches a volume of at least 1 liter,then the culture is switched to perfusion until harvest. In oneembodiment the culture is perfused throughout expansion until harvest.In one embodiment during expansion the perfusion rate is at least onebioreactor bag volume per day. In one embodiment the perfusion rate isless than one bioreactor bag volume per day. In one embodiment theperfusion rate is greater than one bioreactor bag volume per day.

In one embodiment the volume of the culture media in bioreactor isincrementally increased to 1000 ml during expansion to maintain a celldensity of about 2E6 to about 4E6 nucleated cells/ml. In one embodimentthe bioreactor volume is increased from at least 200 ml to at least 1000ml. In one embodiment the bioreactor volume is increased from at least300 ml to at least 1000 ml. In one embodiment the bioreactor volume isincreased from at least 400 ml to at least 1000 ml. In one embodimentthe bioreactor volume is increased from at least 500 ml to at least 1000ml. In one embodiment the bioreactor volume is increased from at least600 ml to at least 1000 ml. In one embodiment the bioreactor volume isincreased from at least 700 ml to at least 1000 ml. In one embodimentthe bioreactor volume is increased from at least 800 ml to at least 1000ml. In one embodiment the bioreactor volume is increased from at least900 ml to at least 1000 ml. In one embodiment the bioreactor volume isincreased from at least 200 ml to at least 900 ml. In one embodiment thebioreactor volume is increased from at least 300 ml to at least 900 ml.In one embodiment the bioreactor volume is increased from at least 400ml to at least 900 ml. In one embodiment the bioreactor volume isincreased from at least 500 ml to at least 900 ml. In one embodiment thebioreactor volume is increased from at least 600 ml to at least 900 ml.In one embodiment the bioreactor volume is increased from at least 700ml to at least 900 ml. In one embodiment the bioreactor volume isincreased from at least 800 ml to at least 900 ml. In one embodiment thebioreactor volume is increased from at least 200 ml to at least 800 ml.In one embodiment the bioreactor volume is increased from at least 300ml to at least 800 ml. In one embodiment the bioreactor volume isincreased from at least 400 ml to at least 800 ml. In one embodiment thebioreactor volume is increased from at least 500 ml to at least 800 ml.In one embodiment the bioreactor volume is increased from at least 700ml to at least 800 ml. In one embodiment the bioreactor volume isincreased from at least 700 ml to at least 800 ml. In one embodiment thebioreactor volume is increased from at least 300 ml to at least 700 ml.In one embodiment the bioreactor volume is increased from at least 200ml to at least 800 ml. In one embodiment the bioreactor volume isincreased from at least 400 ml to at least 800 ml. In one embodiment thebioreactor volume is increased from at least 500 ml to at least 800 ml.In one embodiment the bioreactor volume is increased from at least 600ml to at least 800 ml. In one embodiment the bioreactor volume isincreased from at least 700 ml to at least 800 ml. In one embodiment thebioreactor volume is increased from at least 200 ml to at least 700 ml.In one embodiment the bioreactor volume is increased from at least 200ml to at least 700 ml. In one embodiment the bioreactor volume isincreased from at least 300 ml to at least 700 ml. In one embodiment thebioreactor volume is increased from at least 400 ml to at least 700 ml.In one embodiment the bioreactor volume is increased from at least 500ml to at least 700 ml. In one embodiment the bioreactor volume isincreased from at least 600 ml to at least 700 ml. In one embodiment thebioreactor volume is increased from at least 200 ml to at least 600 ml.In one embodiment the bioreactor volume is increased from at least 300ml to at least 600 ml. In one embodiment the bioreactor volume isincreased from at least 400 ml to at least 600 ml. In one embodiment thebioreactor volume is increased from at least 500 ml to at least 600 ml.In one embodiment the bioreactor volume is increased from at least 200ml to at least 500 ml. In one embodiment the bioreactor volume isincreased from at least 300 ml to at least 500 ml. In one embodiment thebioreactor volume is increased from at least 400 ml to at least 500 ml.In one embodiment the bioreactor volume is increased from at least 200ml to at least 400 ml. In one embodiment the bioreactor volume isincreased from at least 300 ml to at least 400 ml. In one embodiment thebioreactor volume is at least 200 ml, 250 ml, 300 ml, 350 ml, 400 ml,450 ml, at least 500 ml, at least 550 ml, at least 600 ml, at least 650ml, at least 700 ml, at least 750 ml, at least 800 ml, at least 850 ml,at least 900 ml, at least 950 ml, at least 1000 ml, or above 1000 ml. Inone embodiment the bioreactor volume is at least 200 ml. In oneembodiment the bioreactor volume is at least 300 ml. In one embodimentthe bioreactor volume is at least 400 ml. In one embodiment thebioreactor volume is at least 500 ml. In one embodiment the bioreactorvolume is at least 600 ml. In one embodiment the bioreactor volume is atleast 700 ml. In one embodiment the bioreactor volume is at least 800ml. In one embodiment the bioreactor volume is at least 900 ml. In oneembodiment the bioreactor volume is at least 1000 ml. In one embodimentthe bioreactor volume is greater than 1000 ml.

In one embodiment, as the culture volume and/or cell density increases,the rocking rate increases. In one embodiment the rocking rate increasesfrom at least 2 RMP to at least 3 RPM. In one embodiment the rockingrate increases from at least 2 RPM to at least 4 RMP. In one embodimentthe rocking rate increases from at least 2 RPM to at least 5 RMP. In oneembodiment the rocking rate increases from at least 2 RPM to at least 6RMP. In one embodiment the rocking rate increases from at least 3 RPM toat least 4 RMP. In one embodiment the rocking rate increases from atleast 3 RPM to at least 5 RMP. In one embodiment the rocking rateincreases from at least 3 RPM to at least 6 RMP. In one embodiment therocking rate increases from at least 4 RPM to at least 5 RMP. In oneembodiment the rocking rate increases from at least 4 RPM to at least 6RMP. In one embodiment the rocking rate increases from at least 5 RPM toat least 6 RMP. In one embodiment the rocking rate is at least 2 RPM ata 2° angle. In one embodiment the rocking rate is at least 3 RPM at a 3°angle. In one embodiment the rocking rate is at least 4 RPM at a 4°angle. In one embodiment the rocking rate is at least 5 RPM at a 5°angle. In one embodiment the rocking rate is at least 6 RPM at a 6°angle.

In one embodiment, as the volume of the culture media is incrementallyincreased to 1000 ml, the rocking rate is incrementally increased to 6RPM. In one embodiment the volume of the culture media in the bioreactorbag at the start of expansion is about 300 ml at a rocking rate of 2 rpmat a 2° angle maintained at a perfusion rate of one bioreactor bagvolume a day. In one embodiment when the culture reaches a cell densityof 4E6 nucleated cells/ml, the volume of the culture media in thebioreactor bag is increased to 600 ml with a perfusion rate of onebioreactor bag volume per day at a rocking rate of 4 rpm at a 4° angle.In a related embodiment when the culture reaches a density of 4E6nucleated cells/ml, the volume of the culture media in the closed singleuse bioreactor bag is increased to 1000 ml with perfusion rate of onebioreactor bag volume per day, at a rocking rate of 6 rpm at a 6° angle.

In one embodiment, the culture in the single use bioreactor bag ismaintained at 80-100% O₂.

In one embodiment the cells are expanded for 7 to 14 days. In oneembodiment the cells are expanded for 7 to 13 days. In one embodimentthe cells are expanded for 7 to 12 days. In one embodiment the cells areexpanded for 7 to 11 days. In one embodiment the cells are expanded for7 to 10 days. In one embodiment the cells are expanded for 7 to 9 days.In one embodiment the cells are expanded for 7 to 8 days. In oneembodiment the cells are expanded for 8 to 14 days. In one embodimentthe cells are expanded for 8 to 13 days. In one embodiment the cells areexpanded for 8 to 12 days. In one embodiment the cells are expanded for8 to 11 days. In one embodiment the cells are expanded for 8 to 10 days.In one embodiment the cells are expanded for 8 to 9 days. In oneembodiment the cells are expanded for 9 to 14 days. In one embodimentthe cells are expanded for 9 to 13 days. In one embodiment the cells areexpanded for 9 to 12 days. In one embodiment the cells are expanded for9 to 11 days. In one embodiment the cells are expanded for 9 to 10 days.In one embodiment the cells are expanded for 10 to 14 days. In oneembodiment the cells are expanded for 10 to 13 days. In one embodimentthe cells are expanded for 10 to 12 days. In one embodiment the cellsare expanded for 10 to 11 days. In one embodiment the cells are expandedfor 11 to 14 days. In one embodiment the cells are expanded for 11 to 13days. In one embodiment the cells are expanded for 11 to 12 days. In oneembodiment the cells are expanded for 12 to 14 days. In one embodimentthe cells are expanded for 12 to 13 days. In one embodiment the cellsare expanded for 13 to 14 days. In one embodiment the cells are expandedfor 7, 8, 9, 10, 11, 12, 13, 14, or more days. In one embodiment thecells are expanded for at least 7 days. In one embodiment the cells areexpanded for at least 8 days. In one embodiment the cells are expandedfor at least 9 days. In one embodiment the cells are expanded for atleast 10 days. In one embodiment the cells are expanded for at least 11days. In one embodiment the cells are expanded for at least 12 days. Inone embodiment the cells are expanded for at least 13 days. In oneembodiment the cells are expanded for at least 14 days.

In one embodiment, the cells are expanded at 34-39° C. In oneembodiment, the cells are expanded at 34-35° C. In one embodiment, thecells are expanded at 35-37° C. In one embodiment, the cells areexpanded at 35-36° C. In one embodiment, the cells are expanded at36-37° C. In one embodiment, the cells are expanded at 34° C., 35° C.,36° C., or 37° C. In one embodiment, the cells are expanded at 34° C. Inone embodiment, the cells are expanded at 35° C. In one embodiment, thecells are expanded at 36° C. In one embodiment, the cells are expandedat performed at 37° C.

“Engineered T cells” and “genetically engineered T cells”, are usedinterchangeable and refer to T cells that have been modified by theintroduction of extra genetic material, in particular, polynucleotidesencoding proteins of interest, such as receptors expressed on a cellsurface. Examples of cell surface receptors include chimeric antigenreceptors (CAR) and T-cell receptors (TCR). The terms “isolatedengineered T cell” and “isolated genetically engineered T cells”specifically refer to T cells that have been modified or manipulated,such as genetic modification, or not normally found in nature. As usedherein, T cell or T lymphocyte refers to a type lymphocyte that activelyparticipates in the body's immune response.

The terms “polynucleotide” and “nucleic acid molecule” are usedinterchangeably throughout and include both single-stranded anddouble-stranded nucleic acids and includes genomic DNA, RNA, mRNA, cDNA,or synthetic origin or some combination thereof which is not associatedwith sequences normally found in nature. The terms “isolatedpolynucleotide” or “isolated nucleic acid molecule” specifically referto sequences of synthetic origin or those not normally found in nature.Isolated nucleic acid molecules comprising specified sequences mayinclude, in addition to the specified sequences, coding sequences for upto ten or even up to twenty other proteins or portions thereof or mayinclude operably linked regulatory sequences that control expression ofthe coding region of the recited nucleic acid sequences, and/or mayinclude vector sequences. The nucleotides comprising the nucleic acidmolecules can be ribonucleotides or deoxyribonucleotides or a modifiedform of either type of nucleotide. The modifications include basemodifications such as bromouridine and inosine derivatives, ribosemodifications such as 2′,3′-dideoxyribose, and internucleotide linkagemodifications such as phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,phoshoraniladate and phosphoroamidate.

The terms “polypeptide” or “protein” are used interchangeably throughoutand refer to a molecule comprising two or more amino acid residuesjoined to each other by peptide bonds. Polypeptides and proteins alsoinclude macromolecules having one or more deletions from, insertions to,and/or substitutions of the amino acid residues of the native sequence,that is, a polypeptide or protein produced by a naturally-occurring andnon-recombinant cell; or is produced by a genetically-engineered orrecombinant cell, and comprise molecules having one or more deletionsfrom, insertions to, and/or substitutions of the amino acid residues ofthe amino acid sequence of the native protein. Polypeptides and proteinsalso include amino acid polymers in which one or more amino acids arechemical analogs of a corresponding naturally-occurring amino acid andpolymers. Polypeptides and proteins are also inclusive of modificationsincluding, but not limited to, glycosylation, lipid attachment,sulfation, gamma-carboxylation of glutamic acid residues, hydroxylationand ADP-ribosylation. Polypeptides and proteins of interest can be ofscientific or commercial interest, including those useful as componentsof cell therapy therapeutics. Proteins of interest include, among otherthings, membrane-bound proteins.

The invention provides a method for producing engineered T cells thatexpress a protein of interest. In one embodiment the protein of interestis a cell surface receptor. The cell surface receptor is a geneticallyengineered receptors such as T cell receptors (TCRs) and chimericantigen receptors (CARs or CAR-T cells, TRUCKs (chimeric antigenreceptors that redirect T cells for universal cytokine-mediatedkilling), and armored CARs (designed to modulate an immunosuppressiveenvironment)) and as well as other proteins comprising an antigenbinding molecule that interacts with that targeted antigen. Theseengineered receptors are expressed on the surface of the T cell. In oneembodiment the cell surface receptor is a T cell receptor or a chimericantigen receptor.

TCRs are membrane anchored heterodimer protein complexes comprising an achain and a β chain complexed with the invariant CD3 chain molecules.The variable domain of the TCR α- and β-chains each have threecomplementarity-determining regions (CDRs) that recognize a peptidederived from the protein presented by/bound within the groove of anMHC/HLA molecule, and a constant domain that engages in disulfidebonding to link the chains together. CD3 and zeta activate the Tlymphocyte through signal transduction. The TCR have the potential torecognize antigens presented on the surface of numerous cells, such ascancer cells, inflammatory cells and cells from other sources.

CARs are engineered transmembrane proteins, comprising an extracellulardomain, typically comprising an antigen binding protein or domain thatbinds to an antigen of interest, a hinge region, a transmembrane domain,and an intracellular (cytoplasmic) domain.

The extracellular domain may be derived either from a synthetic or froma natural source, such as the proteins described herein. Theextracellular domains often comprise a hinge portion, sometimes referredto as a “spacer” region. Hinges may be derived from the proteins asdescribed herein, particularly the c0-stimulatory proteins describedherein, as well as immunoglobulin (Ig) sequences or other suitablemolecules to achieve the desired special distance from the target cell.

A transmembrane domain may be fused to the extracellular orintracellular domain of the CAR. The transmembrane domain may be derivedeither from a synthetic or from a natural source, such as the proteinsdescribed herein, particularly the costimulatory proteins describedherein.

An intracellular (cytoplasmic) domain may be fused to the transmembranedomain and can provide activation of at least one of the normal effectorfunctions of the immune cell. Effector function includes cytolyticactivity or helper activity including the secretion of cytokines.Intracellular domains can be derived from the proteins described herein,particularly from CD3.

CARs can be engineered to bind to an antigen (such as a cell-surfaceantigen) by incorporating an antigen binding molecule that interactswith that targeted antigen. CARs typically incorporate an antigenbinding domain (such as scFv) in tandem with one or more costimulatory(“signaling”) domains and one or more activating domains.

In one embodiment the antigen binding molecule is an antibody fragmentthereof, and more preferably one or more single chain antibody fragment(“scFv”). scFvs are preferred for use in CARs because they can beengineered to be expressed as part of a single chain along with theother CAR components. See Krause et al., J. Exp. Med., 188(4): 619-626,1998; Finney et al., Journal of Immunology, 161: 2791-2797, 1998.

CARs incorporate one or more costimulatory (signaling) domains toincrease their potency. See U.S. Pat. Nos. 7,741,465, and 6,319,494, aswell as Krause et al. and Finney et al. (supra), Song et al., Blood119:696-706 (2012); Kalos et al., Sci Transl. Med. 3:95 (2011); Porteret al., N. Engl. J. Med. 365:725-33 (2011), and Gross et al., Annu. Rev.Pharmacol. Toxicol. 56:59-83 (2016). Suitable costimulatory domains canbe derived from, among other sources, CD28, CD28T, OX40, 4-1BB/CD137,CD2, CD3 (alpha, beta, delta, epsilon, gamma, zeta), CD4, CD5, CD7, CD8,CD9, CD16, CD22, CD27, CD30, CD 33, CD37, CD40, CD 45, CD64, CD80, CD86,CD134, CD137, CD154, PD-1, ICOS, lymphocyte function-associatedantigen-1 (LFA-1 (CD11a/CD18), CD247, CD276 (B7-H3), LIGHT (tumornecrosis factor superfamily member 14; TNFSF14), NKG2C, Ig alpha(CD79a), DAP-10, Fc gamma receptor, MHC class I molecule, TNF, TNFr,integrin, signaling lymphocytic activation molecule, BTLA, Toll ligandreceptor, ICAM-1, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR),KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha,CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a,ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1-1d, ITGAE, CD103,ITGAL, CD1-la, LFA-1, ITGAM, CD1-1b, ITGAX, CD1-1c, ITGB1, CD29, ITGB2,CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4(CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160(BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT,41-BB, GADS, SLP-76, PAG/Cbp, CD19a, CD83 ligand, or fragments orcombinations thereof. The costimulatory domain can comprise one or moreextracellular portions, a transmembrane portion, and an intracellularportion.

CARs also include one or more activating domains CD3 zeta is an elementof the T cell receptor on native T cells and has been shown to be animportant intracellular activating element in CARs.

Cell surface molecules can be targeted with a traditional CAR.Intracellular molecules can be targeted with TCRs that recognize apeptide derived from the protein presented by or bound within the grooveof an MHC/HLA molecule. Such targets include alpha folate receptor, 5T4,AFP, ADAM 17, 17-A, ART-4, α_(v)β₆integrin, BAGE. Bcr-abl, BCMA, B7-H3,B7-H6, CAIX, CAMEL, CAP-1, Carbonic anhydrase IX, CASP-8, CDC27m, CD19,CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7/8, CD70 (CD27L or TNFSF7),CD79a, CD79b, CD123, CD138, CD171, CDK4/m, cadherin 19 (CDH19),Placental-Cadherin (CDH3), CEA, CLL-1, CSPG4, CT, Cyp-B, DAM, DDL3, EBV,EGFR, EGFRvIII, EGP2, EGP40, ELF2M, ErbB2 (HER2), EPCAM, EphA2, EpCAM,ETV6-AML1, FAP, fetal AchR, FLT3, FRa, G250, GAGE, GD2, GD3, ‘Glypican-3(GPC3), GNT-V, GP-100, HAGE, HBV, HCV, HER-2/neu, HLA-A, HPV, HSP70,HST-2, hTERT, iCE, IgE, IL-11Ra, IL-13Ra2, Kappa, KIAA0205, LAGE,Lambda, LDLR/FUT, Lewis-Y, MAGE, MAGE1, MAGEB2, MART-1, /Melan-A, MC1R,MCSP, MUM-1, MUM-2, MUM-3, mesothelin (MSLN), Mucl, Muc16, Myosin/m,NA88-A, NCAM, NKG2D Ligands, NY-ESO-1, P15, p190 minor bcr-abl,PML/RARa, PRAME, PSA, PSCA, PSMA, RAGE, ROR1, RU1, RU2, SAGE, SART,SSX-1, SSX-2, SSX-3, Survivin, TAA, TAG72, TEL/AML1, TEMs, TPI, TRP-1,TRP-2, TRP-2/INT2, VEGFR2 and WT1. See also, Löffler et al., Blood85(6): 2098-2103, 15 Mar. 2000; Wolf et al., Drug Discovery Today 10(8):1237-1244, September 2005; Baeuerle & Reinhardt, Cancer Res 69(12):4941-4944, Jun. 15, 2009; Baeuerle et al., Curr. Opin, Mol. Ther. 11:22-30, 2009; Nagorsen & Baeuele, Exp Cell Res 317:1255-1260, 2011;Nagorsen et al., Pharmacology & Therapeutics 136: 334-342, 2012; Huehlset al., Immunology and Cell Biology 93:290-296, 2015; and Stieglmaier etal., Expert Opin Biol Ther 15(8): 1093-1099, 2015.

The process could be used for producing T cells expressing the CD19CARs, such as Tisagenlecleucel (Kymriah™) and axicabtagene ciloleucel(Yescarta®).

In one embodiment the cell surface receptor recognizes an antigenictarget associated with a target cell such as a cell surface molecule oran intermolecular molecule. The target cell is any cell that presents anantigen that is recognized by the gene of interest. In one embodimentthe target cell is a cancer cell. A cancer cell is a cell associatedwith a cancer, including cells associated with metastatic tumors, solidtumors, blood cancers, brain cancers, breast cancers, colon cancers,kidney cancers, lung cancers, liver cancers, ovarian cancers, pancreaticcancers, prostate cancers, skin cancers, spleen cancers, stomachcancers, thyroid cancers.

Greater transduction efficiency allows for delivering a smaller, moreeffective dose to the patient. When fewer T cells transduced with thegene of interest are produced there are fewer genetically engineered Tcells available for administration to the patient resulting in a lesseffective dose. The method described herein achieves a high transductionrate (high transgene expression greater than 40% on CD8 T cells) at alower lentiviral vector MOI. Less lentiviral vector is required to makemore engineered T cells expressing the protein of interest, the methodis more efficient and cost effective. The invention provides a methodfor increasing the transgene expression in genetically engineeredautologous T cells expressing a protein of interest, the methodcomprising inoculating a closed single use bioreactor bag containingculture media with apheresed donor cells and one or more soluble T cellactivators, wherein at least one soluble T-cell activator is bound to atleast one donor cell at the time of inoculation and the bioreactor bagis part of a rocking bioreactor platform, culturing the cells in theclosed single use bioreactor bag continuously rocking at a rate of about2 RPM, transducing the cells in the closed single use bioreactor bagwith at least one soluble viral vector comprising a polynucleotide whichencodes the protein of interest continuously rocking at a rate of about2 RPM, and expanding the cells in the closed single use bioreactor bagat a rocking rate of about 2 RPM and increasing the culture volume androcking the rate as needed to maintain the culture until harvest,wherein the transgene expression is greater than the transgeneexpression of genetically engineered autologous T cells derived from anenriched population of T cells from the same apheresed donor cells andexpressing the same protein of interest.

Genetically engineered T cells are recovered by harvesting when the celldensity has reached a desired or predetermined state or at apredetermined or desired time point. The genetically engineered T cellsmay be recovered from the bioreactor using methods known in the art,such as separation using gravity or centrifugation. The cells may alsobe recovered using semiautomated and fully automated cell separationsystems, including blood separation systems. Such systems arecommercially available and include Sepax® 2 and Sefia™ S-2000 CellProcessing Systems (GE Healthcare), Cobe 2991 cell processor (TerumoBCT, Lakewood, Colo., CellSaver 5 (Haemonetics, (Boston, Mass.), and thelike. The recovered cells are typically washed and concentrated into asuitable formulation for administration to the patient and forcryopreservation.

With the method described herein, yields of greater than 10 billion witha viability of greater than 90% are seen in a 10 day process. Yieldsgreater than 20 billion were seen after when cell expansion was extendedfor several more days.

Samples may be taken during expansion and/or at harvest for qualitycontrol testing. Such QC tests may include those that quantify thetransduction efficiency of the genetically engineered T cells such asdetermining % TCR on cell surface using flow cytometry and determiningvector copy number using qPCR to measure integration. Viability can alsobe tested before releasing the product to be delivered to the patient.

Standard procedures known in the art may be used for cryopreservation ofrecovered genetically engineered T cells for storage and/or preparationfor use in a human subject. In one embodiment the recovered cells areeluted at 2E8 cells/ml in saline supplemented with 1% HSA and werefurther formulated at a 1:1 ratio with Hyclone cryopreservation media(GE Healthcare) supplemented with 5% human serum albumin.

A fraction of the formulated engineered T cells, as well as anyapheresed cells not used in the method, can be cryopreserved by methodsknown in the art to provide a permanent source of such cells for thefuture treatment of the patient.

When needed for treatment, the cryopreserved genetically engineered Tcells are thawed and diluted into a saline buffer or other suitablemedia for administration to the donor by infusion in atreatment-effective amount. Suitable infusion media can be any isotonicmedium formulation, typically normal saline, Normosol™ R (Abbott) orPlasma-Lyte™ A (Baxter), but also 5% dextrose in water or Ringer'slactate can be utilized. The infusion medium can be supplemented withhuman serum albumin. The volume is typically kept minimal as possible, apharmaceutically acceptable dose is based on the number and viability ofthe cells, the indication to be treated and the patient. Therapeuticdoses are typically between a few million and a few billion cells,depending on the patient's condition and need, among other factors. Anexemplary dose may be ≤100 ml at about 100E6 cells/ml.

The invention provides a method of treating a patient with geneticallyengineered autologous T cells expressing a protein of interestcomprising, incubating apheresed cells from the patient with one or moreT cell activators selected from the group consisting of an anti CD3antibody, an anti CD2 antibody, and an anti CD28 antibody or bindingfragments thereof, to allow for saturation of antibody binding,inoculating a closed single use bioreactor bag containing culture mediawith the apheresed cells, wherein the bioreactor bag is part of arocking bioreactor platform, culturing the cells in the closed singleuse bioreactor bag continuously rocking at a rate of about 2 RPM,transducing the cells in the closed single use bioreactor bag with atleast one soluble viral vector comprising a polynucleotide which encodesthe protein of interest continuously rocking at a rate of about 2 RPM,and expanding the cells in the closed single use bioreactor bag at arocking rate of about 2 RPM, increasing the culture volume and rockingthe rate as needed to maintain the culture at a desired cell densityuntil harvest, harvesting and formulating the cells forcryopreservation, freezing the cells and storing until needed foradministering to the patient, thawing and resuspending the cells in asuitable media for infusion, and reintroducing a pharmaceuticallyeffective amount of the genetically engineered autologous T cellsexpressing the protein of interest into the patient.

The formulated engineered T cells may be administered either alone, oras a pharmaceutical composition in combination with diluents and/or withother components such as IL-2 or other cytokines or cell populations.

Methods are provided for using the engineered T cells for treatingconditions, indications, diseases, disorders and the like. The T cellexpresses a gene of interest, such as a cell surface receptor,recognizes an antigenic target associated with a target cell that isassociated with the condition, indication, disease, disorder or thelike. Such conditions, diseases or disorders including cancers, tumors,solid tumors, hematologic disorders, leukemia, lymphomas, viralinfections, inflammatory disease or disorders, and/or autoimmune diseaseor disorders. In one embodiment of the invention, the geneticallyengineered T cells are used to treat an indication in a subject. In oneembodiment of the invention, genetically engineered T cells are used totreat a cancer patient. In some embodiments, the invention providescreating a T cell-mediated immune response in a donor, comprisingadministering to the donor an effective amount of an engineeredautologous T cell that expresses a protein of interest. In someembodiments, the T cell-mediated immune response is directed against atarget cell or cells. In some embodiments, the engineered autologous Tcell comprises a genetic construct expressing one or more chimericantigen receptors, T cell receptors and/or other proteins of interest.In some embodiments, the target cell is a tumor cell. In someembodiments, the invention comprises a method for treating or preventingan indication, said method comprising administering to a subject in needthereof an effective amount of a genetically engineered autologous Tcell made by the method described herein. In some aspects, the inventioncomprises a method for treating or preventing inflammatory and/orautoimmune disorders.

While the terminology used in this application is standard within theart, definitions of certain terms are provided herein to assure clarityand definiteness to the meaning of the claims. Units, prefixes, andsymbols may be denoted in their SI accepted form. Numeric ranges recitedherein are inclusive of the numbers defining the range and include andare supportive of each integer within the defined range. The methods andtechniques described herein are generally performed according toconventional methods well known in the art and as described in variousgeneral and more specific references that are cited and discussedthroughout the present specification unless otherwise indicated. Alldocuments, or portions of documents, cited in this application,including but not limited to patents, patent applications, articles,books, and treatises, are hereby expressly incorporated by reference.

The present invention is not to be limited in scope by the specificembodiments described herein that are intended as single illustrationsof individual aspects of the invention, and functionally equivalentmethods and components are within the scope of the invention. Indeed,various modifications of the invention, in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and accompanying drawings. Such modificationsare intended to fall within the scope of the appended claims. What isdescribed in an aspect or embodiment of the invention can be combinedwith other aspects and/or embodiments of the invention.

EXAMPLES Example 1 Comparison Study of Three Processes

1: Autologous T cells generated with the closed continuous autologous Tcell bioprocessing method where activation to expansion is conducted ina Xuri W25 Cell Expansion System. “Xuri W25 bioreactor”.

2: A bioprocessing system where activation to expansion is conducted inG-Rex® 6 and 24 well plates. “G-Rex”.

3: Hybrid bioprocessing system where activation and transduction areconducted in a gas permeable bag followed by T cell expansion in a XuriW25 Cell Expansion System. “Permeable bag”.

In addition, two different culture media supplemented with IL2 (Media 1)or TWS119+IL7+IL21 (Media 2), were also compared using the closedcontinuous autologous T cell bioprocessing system and the G-Rex® plates.

T cell production was evaluated for its purity, phenotype, and in vitroproperties.

On day 0, a fresh Leukopack (Hemacare, Northridge, Calif.), containing300 ml fresh leukapheresis product collected from normal peripheralblood was sterile welded to a CD-600.1 Sepax Cell Separation Kit (GE)and processed under the Culture Wash-Pro program. The Leukopack®contained leukocytes, erythrocytes, and platelets. The cells were washedin 1 L ClinMACS PBS/EDTA, 5 ml human serum albumin (Miltenyi Biotec, SanDiego, Calif.) to remove plasma and any apheresis buffers using a SempaxCPro Cell Separation System equipped with a CS-600.1 Kit (GEHealthcare), according to manufacturer's instructions. Based on theinitial white blood cell (WBC) count indicated on the donor informationsheet accompanying the Leukopack®, the cells were eluted at a celldensity of 150E6 WBC/ml with ˜50 ml OpTimizer complete media.

The nucleated cells in the washed leukapheresed harvest sample werecounted using NC200™ Automated Cell Counter ChemMetec, Denmark) andfollowed by determining total viable cell count, viability, andimmunophenotyping of the washed cells.

1) Closed Continuous Autologous T Cell Bioprocess Using the a Xuri W25Cell Expansion System. “Xuri W25 Bioreactor”

A portion of the washed apheresed donor cells comprising 1.2E9 nucleatedcells was then transferred into two transfer bags (Charter Medicine,Winston-Salem, N.C.) under the Dilute program using the Sepax C-Proprocessing system with the same CD-600.1 Kit. In to one bag was addedabout 8 ml of Media 1: (OpTmizer complete media containing 300 IU/mLrhIL2 (ThermoFisher)). In to the second bag was added about 8 ml ofMedia 2: (OpTmizer complete media containing 5 uM TWS119 (CaymanChemical, Ann Arbor, Mich.), 20 ng/ml IL7 (Stemcell Technologies,Cambridge, Mass.) and 20 ng/ml IL-21 (Stemcell Technologies)). Each bagwas incubated with 7.5 ml ImmunoCult™ Human CD3/CD28/CD2 (25 μl/ml ofcells, Stemcell Technologies) at room temperature for 1 hour to allowsaturation of antibody binding. The concentration of the Immunocultadded was determined on a final activation culture volume of 300 ml asdescribed in the step below. OpTmizer complete media: OpTimizer T cellexpansion medium (ThermoFisher, Waltham, Mass.), 2.5% Immune Cell SRsupplement (ThermoFisher), 2.6% CTS T cell supplement (ThermoFisher), 1%GlutaMax (ThermoFisher), 0.1% Pluronic F68 and 300 IU/ml IL2.

A 2 L Xuri SP Perf Cellbag bioreactor (GE Healthcare) was connected to aXuri Cell Expansion System W25 and 300 ml Media 1 was added and allowedto equilibrate at 37° C., 5% CO₂, gas flow rate of 0.1 L/min, and arocking rate of 6 rpm at a 6° angle. A second 2 L Xuri SP Perf Cellbagbioreactor was also connected to a Xuri Cell Expansion System W25 and300 ml Media 2 was similarly equilibrated.

The transfer bags containing the cells in Media 1 and Media 2 weresterile welded to the Xuri Cellbag feed-lines and the contentstransferred into the bags by gravity. The cells were then culturedovernight at 37° C., 5% CO₂, gas flow rate 0.1 L/min at a rocking rateof 2 rpm at a 2° angle to allow for the activation of T cells.

On Day 1, the nucleated cells in each bag were counted. An amount of alentiviral vector (comprising a polynucleotide encoding a TCR) thatcorresponded to a MOI of 1 functional titer was diluted in 10 ml ofMedia 1 or Media 2 and placed in transfer bags. The transfer bags weresterile welded on to the Xuri Cellbag feed-lines and lentivirus wastransferred into each of the bioreactors via gravity flow. The cellswere incubated at 37° C., 5% CO₂, gas flow rate of 0.1 L/min, and arocking rate of 2 rpm at a 2° angle, for 20-24 hours.

On Day 2, about half the volume of the culture media in each bioreactorbag was exchanged using three 50 ml bag washouts through feed-line andwaste-line. Cell counts were taken daily and viable cell density (VCD)and viability were determined using NC200™ Automated Cell Counter(ChemMetec), dissolved oxygen and metabolites were also measured. Cellphenotypes were determined for all samples tested. The bags weremaintained on the rocking bioreactors at 2 rpm at a 2° angle, 37° C., 5%CO₂, gas flow rate of 0.1 L/min, for 24 hours.

On Days 3-7, additional OpTimizer complete media containing either theIL-2 (Media 1) or TWS119/IL17/IL21 (Media 2), was added by fedbatch/perfusion feeds to a final volume of 1 L to both cultures, tomaintain the cell density above 2E6 cells/ml. As volume increased, therocking speed and angle was increase to 4 RPM at a 4° angle, to maintaina cell density at the desired level.

Total viable cell density, viability, glucose and lactate measurementswere taken each day. Phenotyping was determined on days 3, 5 and 7.

On Days 7-10, both cultures were switched to perfusion feeding at a rateof one bag volume/day. Both bioreactor bags were now fed with Media 1.As cell density increased, dissolved 02 levels were maintained at 80% byfeedback control.

Total viable cell density, viability, glucose, and lactate measurementswere made each day. Phenotyping was done on day 10.

The cells were harvested on Day 10. The Xuri SP Per bioreactor bags weresterile welded to a Selfia 5200 Cell Processing System using theFlexCell program and CT-800.1 Cell Processing kit (GE Healthcare. The Tcells were concentrated to −20 ml at a 75 ml/min flow rate. One washcycle was performed using 0.9% saline (Baxter, Deerfield, Ill.)supplemented with 1% vol human serum albumin (HSA). The wash wasperformed at 380×g for 5 min.

Cells were reconstituted in equal volume saline plus 1% HSA plusHyClone™ Cryopreservation Media (GE Healthcare) supplemented with 5% HSAand cryopreserved by freezing to −80° C. using the control rate freezerVia Freeze (GE Healthcare) and storing in liquid nitrogen.

2. G-Rex Plates “G-Rex”.

The cells were cultured in 24-well or 6-well G-Rex (WilsonWolf, NewBrighton, Minn.) plates following manufacturer's protocols, using Media1 and Media 2. 1E6 cells/well were seeded in the G-Rex® plates andactivated with ImmunoCult™ Human CD3/CD28/CD2 (25 ug/ml media withcells) on day 0. Approximately 24 hours after activation, cells wereseeded at 1E6 cells/well (concentration 2E6 cells/mL) and an amount ofthe same lentiviral vector (comprising a polynucleotide encoding a TCR),corresponding to a MOI of 1 functional titer was added to each well. Onday 2, Media 1 and Media 2 was added to the full capacity of the wells(total 6 mL) to dilute the virus. The media was changed, and the cellswere fed every 2-3 days.

Cell counts were taken daily and viable cell density (VCD) and viabilitywere determined using a NC-200™ Automated Cell Counter (ChemoMetec),dissolved oxygen and metabolites were also measured. Cell phenotypeswere determined for all samples tested.

3. Hybrid Bioprocessing System, Activation and Transduction Conducted ina Gas Permeable Bag Followed by T Cell Expansion in a Xuri W25 CellExpansion System. “Perfusion Bag”.

Washed apheresed donor cells comprising 500E6 nucleated cells wastransferred into a gas permeable bag (OriGen, Austin, Tex.) with 6 mlImmunoCult™ Human CD3/CD28/CD2 and incubated at 37° C., 5% CO₂ for 24hours.

The nucleated cells were counted. An amount of the same lentiviralvector (comprising a polynucleotide encoding aTCR), corresponding to aMOI of 1 functional titer was added directly to the bag using a syringeand incubated overnight at 37° C., 5% CO₂.

The cells were then split into two gas permeable bags the following dayand were incubated in the bags at 37° C., 5% CO₂, until a total of 1billion nucleated cells.

The cells were then inoculated into 2 L Xuri SP Perf Cellbag bioreactorbags on a Xuri W25 Cell Expansion System (GE Healthcare) containing 300ml OpTimizer complete media containing 300 IU/ml IL2, and rocked at arate of 6 rpm at a 6° angle. The bioreactor was scaled up to a 1 Lvolume 24 hours following inoculation. Fresh media was perfused at 500ml/day for 1 day, followed by 1 L/day until harvest.

Cell counts were taken daily and viable cell density (VCD) and viabilitywere determined using a NC-200™ Automated Cell Counter (ChemoMetec),dissolved oxygen and metabolites were also measured. Cell phenotypeswere determined for all samples tested.

The cells were harvested, concentrated and further formulated at a 1:1ratio with HyClone™ Cryopreservation Media (GE Healthcare) supplementedwith 5% HSA and cryopreserved by freezing to 100° C. using a Via Freeze(GE Healthcare) and storing in liquid nitrogen. Viability and recoveryat harvest were measured.

Results

FIG. 1 Shows the growth curve and TCR expression at harvest for the Tcells produced under Condition 1, the closed continuous autologous Tcell bioprocess using the Xuri W25 bioreactor. FIG. 1A shows the growthcurve of cell expansion in the Xuri W25 bioreactor with mediasupplemented with IL2 only or the cocktail of IL7, IL21, and TWS119.Media with IL2 yielded 16 billion T cells in 10 days while media withIL7, IL21, and TWS119 yielded 12 billion T cells in 10 days. FIG. 1Bshows the viability of cell expanded in the Xuri W25 bioreactor withmedia supplemented with IL2 only or the IL7, IL21, and TWS119 cocktail.Cell viability was above 90% throughout expansion and no difference incell viability was observed between the different media conditions.

FIG. 2 shows T cell transgene expression under different conditions. Allcells were transduced with the same lentiviral vector at a MOI=1 one dayafter activation. The transgene was more highly expressed in the cellstransduced in the Xuri W25 bioreactor compared with cells transduced inthe Permeable bag (Hybrid condition 3) or in the G-Rex system (condition2).

FIG. 3 shows the leukocyte subsets in harvested T cells from thedifferent test conditions. Analysis of human B cells (CD19), monocytes(CD14), NK cells (CD56/16), NKT cells (CD3+CD56+), and T cells(CD3+Cd56−) were performed by immunophenotyping. T cell purity in allconditions were above 98% and no significant difference in thepercentage of non-T cells subsets was seen among the differentconditions.

FIG. 4 shows the percentage of T cells subsets on Day 5 (A) and Day 10(B) under different conditions. Tscm, Tcm, Tem, and Tte subsets wereanalyzed by immunophenotyping based on their expression of CD45RA,CD45RO, CD95, and CCR7. T cells produced in the Xuri W25 bioreactor hada higher percentage of Tscm and Tcm than T cells produced in the G-Rexsystem or Permeable bag on Day 5 and a higher percentage of Tcm on Day10 than T cells that was produced in the G-Rex system, indicating a lessdifferentiated phenotype.

FIG. 5 shows the cytotoxic function of engineered T cells characterizedby their ability in target cell lysis (A), IFN-gamma release in responseto target cells (B), and TNF-alpha release in response to target cells(C). Briefly, harvested T cells and peptide-pulsed T2 cells wereco-cultured together at a ratio of 1:1. The T2 cells were Luciferaseengineered, and luminescence was determined using a Steady-Glo®Luciferase Assay System (Promega, Madison, Wis.). The decrease ofluminescence was used to quantify the cell death of T2 cells as anindicator of the cytotoxicity of engineered T cells. As shown in thefigures, engineered T cells generated in the Xuri W25 bioreactor underthe two media conditions (IL2 or IL7, IL21, TWS119) were both morepotent in killing target cells (A), possibly due to their strongerrelease of IFN-gamma (B) and TNF-alpha (C), than engineered T cellgenerated in the static Permeable bag and the static G-Rex system.

FIG. 6 shows resistance to target cell challenge of engineered T cells,characterized by their level of annexin (A) and expression of exhaustionmarker Tim3 (B) after co-culturing with target cells in vitro ateffector to target (E:T) ratio at 1:1. Externalization ofphosphatidylserine in apoptotic cells was detected using recombinantannexin V conjugated to violet-fluorescent Pacific Blue™ dye. T cellswith effector phenotype are susceptible to activation with target cellchallenge and will become apoptotic after activation, thus a lower levelof annexin should indicate T cells with more memory subsets. As shown inthe figures, engineered T cells generated from the Xuri W25 bioreactorhad lower level of annexin than cells from G-Rex system or Permeable bagin response to target cell challenge, thus are more memoryphenotype-like. (A). In addition, engineered T cells generated in theXuri W25 bioreactor and Permeable bag had lower expression of exhaustionmarker Tim3 compared with cells from G-Rex system (B).

The closed continuous autologous T cell bioprocessing method wascompared with the static G-Rex and hybrid gas permeable bag/Xuri systemin their ability to generate autologous lentiviral engineered T cells.The closed continuous method produced T cells with higher transgeneexpression, higher level of cytokine secretion, and more memory-likephenotype when compared with T cells transduced in the static G-Rexsystem or the static gas permeable bag.

Example 2 Addition of a Glycolysis Inhibitor to the Culture Media

On day 0, a fresh Leukopack (Hemacare, Northridge, Calif.), containing300 ml fresh leukapheresis product collected from normal peripheralblood was sterile welded to a CD-600.1 Sepax Cell Separation Kit (GEHealthcare) and processed under Culture Wash-Pro program. The Leukopack®comprised leukocytes, erythrocytes, and platelets. The cells were washedin 1 L ClinMACS PBS/EDTA, 5 ml human serum albumin (Miltenyi Biotec) toremove plasma and apheresis buffers using a Sempax CPro Cell SeparationSystem equipped with a CS-600.1 Kit (GE Healthcare), according tomanufacturer's instructions. The cells were washed and counted asdescribed above in Example 1.

A portion of the washed apheresed donor cells comprising 1.2E9 nucleatedcells were transferred into two transfer bags under the Dilute programusing Sepax C-Pro processing system with the same CS-600.1 Kit (a newkit was not used). Into one bag was added about 8 ml of Media 1(OpTmizer complete media containing 300 IU/mL rhIL2 (ThermoFisher) and 2mM 2-Deoxy-D-glucose (2-DG) (MilliporeSigma, Burlington, Mass.)). Intothe second bag was added about 8 ml of Media 2 (OpTmizer complete mediacontaining 300 IU/mL rhIL2 (ThermoFisher)). To each was added 7.5 mlImmunocult anti-CD3/CD28/CD12 (25 IU/ml media with cells, StemcellTechnologies). The amount of activator was determined based on theculture volume of 300 ml that was used in the culture/activation stepbelow. The bags were incubated for 1 hour at room temperature to allowfor saturation of antibody binding.

A 2 L Xuri SP Perf Cellbag bioreactor (GE Healthcare) was connected to aXuri Cell Expansion System W25 and 300 ml Media 1 was added and allowedto equilibrate at 37° C., 5% CO₂, gas flow rate of 0.1 L/min, and arocking rate of 6 rpm at a 6° angle. A second 2 L Xuri SP Perf Cellbagbioreactor was also connected to a Xuri Cell Expansion System W25 and300 ml Media 2 was similarly equilibrated.

The transfer bags containing the cells in Media 1 and Media 2 weresterile welded to the Xuri Cellbag feed-lines and the contentstransferred into the bags by gravity. The cells were then incubatedovernight at 37° C., 5% CO₂, gas flow rate 0.1 L/min at a rocking rateof 2 rpm at a 2° angle.

On Day 1, the nucleated cells in each bag were counted and the amount ofa lentiviral vector (comprising a polynucleotide encoding a TCR)corresponding to a MOI of 1 functional titer was diluted in 10 ml ofMedia 1 or Media 2 and placed in transfer bags. The transfer bags weresterile welded on Xuri Cellbag feed-lines and the lentivirus was thentransferred into each of the bioreactors via gravity flow. The cellswere incubated at 37° C., 5% CO₂, gas flow rate of 0.1 L/min, and arocking rate of 2 rpm at a 2° angle, for 20-24 hours.

On Day 2, about half the volume of the culture media in each bioreactorbag was exchanged using three 50 ml bag washouts through feed-line andwaste-line. Cell counts were taken daily and viable cell density (VCD),viability, dissolved oxygen, metabolites were determined as describedabove. Cell phenotypes were determined for all samples tested. The bagswere maintained on the rocking bioreactors at 2 rpm at a 2° angle, 37°C., 5% CO₂, gas flow rate of 0.1 L/min, for 24 hours.

On Days 3-9, additional OpTimizer complete media containing eitherIL-2/2-DG (Media 1) and IL2 (Media 2), were added by fed batch/perfusionfeeds to 1 L, to maintain the cell density above 2E6 cells/ml. As theculture volume increased, the rocking speed and angle were increase to 4RPM at a 4° angle, to maintain a sufficient mass transfer inbioreactors. Media 1 containing 2-DG was perfused out with OpTimizercomplete media containing only IL2 (Media 2) when culture volume reached1 L. In a separate experiment, OpTimizer complete media containing 2-DGwas maintained until harvest.

Total viable cell density, viability, glucose and lactate measurementswere taken each day. Phenotyping was determined on Days 3, 5 and 7.

On Days 9-14, both cultures were switched to 1 L/day perfusion feeding,and rocking was set at 6 RPM at a 6° angle. Total viable cell density,viability, glucose, lactate measurements were made each day. Phenotypingwas done on Day 14.

Total viable cell density, viability, glucose and lactate measurementswere taken each day. Phenotyping was determined on Days 9 and 14.

The cells were harvested on Day 14. The Xuri SP Per bioreactor bags weresterile welded to a Selfia 5200 Cell Processing System using theFlexCell program and CT-800.1 Cell Processing kit (GE Healthcare). Tcells were concentrated to ˜20 ml at a 75 ml/min flow rate. One washcycle was performed using 0.9% saline (Baxter, Deerfield, Ill.)supplemented with 1% vol human serum albumin (HSA). The wash wasperformed at 380×g for 5 min.

Viability and harvest recovery were measured. The cells wereconcentrated and further formulated at a 1:1 ratio with Hyclonesupplemented with 5% human serum albumin and cryopreserved by freezingto −80° C. using a Via Freeze (GE Healthcare) and storing in liquidnitrogen

Results

During antigen stimulation, T cells shift to a glycolytic metabolism tosustain effector function. However, the role of glucose metabolism in Tcell differentiation during in vitro expansion is unclear. It washypothesized in this study that glucose metabolism, in particularglycolysis, supported T cell differentiation into effector phenotypeduring expansion and pharmacological inhibition of glycolysis via 2-DGcould attenuate CD3/CD28/CD2 antigen-mediated differentiation andpromote a memory phenotype. In this experiment, 2-DG was added to cellswith the activators to inhibit glycolysis following T cell activation.2-DG was then either removed later in the expansion phase to allowrestoration of glycolysis to support cell expansion or kept in throughthe entire process until harvest. The engineered T cells derived from2-DG-inhibited T cells differentiation produced more Tscm and Tcmcompared with the T cells derived from the media without 2-DG. Thisresulted in a greater T cell yield, greater transgene expression, and amore memory-like phenotype of the T cells.

FIG. 7 shows the growth curve (A) and viability (B) cells expanded inmedia supplemented with (Media 1) 2-DG and without 2-DG (Media 2). Mediacontaining 2-DG yielded 35 billion T cells in 14 days while mediumcondition without 2-DG yielded 25 billion T cells in 14 days. Overallviability was above 70% with the cells from the 2-DG media having aslightly higher viability than those from non-2-DG media from Day 4 toDay 10. This drop of viability during Days 4-10 was possibly due to celldeath of non-T cell leucocytes. Viability returned to higher than 90%until harvest after the T cell population was enriched to greater than90% in the cultures. With 2-DG checking the brake on T cellsdifferentiating into Tem, less cytotoxic cytokine release associatedwith decreased Tem population may also have contributed to higherviability in the samples containing the 2-DG media.

FIG. 8 shows transgene expression of cells expanded in the mediasupplemented with 2-DG (Media 1) and without 2-DG (Media 2). Transgeneexpression by T cells in 2-DG media was slightly higher than that of Tcells in non-2-DG media (56% vs 54%) and was able to maintain at a highlevel (50.4%) when cell density was as high as 35 billion cells/ml. Incontrast, transgene expression of T cell in non-2-DG media decreased to44.4% at high cell density (25 billion cells/ml).

FIG. 9 shows CD25 expression by cells expanded in the media supplementedwith 2-DG (Media 1) and without 2-DG (Media 2). CD25 expression by Tcells in non-2-DG media showed a stronger response to activation that itexpressed at a higher level than that of T cell in 2-DG media (46% vs32%). Even through CD25 expression by T cells in both conditionssaturated at ±80%. T cells in 2-DG media retained CD25 better at Days 7and 9 than the cells in the non-2-DG media. This may be due to thehigher transgene expression and T cell yield for T cells generated inthe 2-DG media.

FIG. 10 shows glucose (A) and lactate (B) concentrations under thedifferent media conditions. Glucose and lactate were measured usingCedex Bio Analyzer (Roche, Indianapolis, Ind.). Inhibition of glycolysisunder the 2-DG media condition (Media 1) was evidenced by higher glucoseconcentration (lower glucose consumption) (A) and higher lactateconcentration (B) in spent media compared to that from the non-2-DGcondition (Media 2). In addition, the effect of 2-DG was reversible asrestoration of glucose (A) and lactate (B) levels was found afterremoval of 2-DG from the media from Day 7 to 14.

FIG. 11 shows T cell differentiation phenotyping of cells expanded mediasupplemented with (Media 1) and without 2-DG (Media 2). 2-DG was addedto culture medium between Days 0 and 7 and the feed media was replacedwith non-2-DG media (Media 1) from Days 7-14. The T cells in the 2-DGmedia were less differentiated, suggested by higher Tscm subset (26% vs14%) at Day 5 and higher combined Tcm and Tscm (86% vs 56%) at Day 7.The T cells continued differentiation after 2-DG was perfused out fromculture media and ended at a similar level of differentiation as T cellsin non-2-DG media (Media 1).

FIG. 12 shows leukocyte subsets in harvested T cells under the differentmedia conditions. Analysis of human B cells (CD19), monocytes (CD14), NKcells (CD56/16), NKT cells (CD3+CD56+), and T cells (CD3+Cd56−) wereperformed by immunophenotyping. Although NKT cells expanded from 5% to±10%, CD3+ cell purity in both conditions was above 98% and nosignificant difference in the percentage of non-T cells subsets werefound between 2-DG and non-2-DG media conditions.

FIG. 13 shows the results from potency assays of engineered TCR T cells.T2 cells expressing Luciferase were pulsed with the peptide recognizedby the engineered TCR or with DMSO for 4 hours and the cells were washedbefore setting up a co-culture assay with 20,000 total T cells at a 1:1(E:T) ratio. (A) 48 hours later, the luciferase signal was measuredusing the Steady Glo® Luciferase Assay System (Promega). Specificcytotoxicity was measured relative to the no peptide T2 cells culturedwith the TCR T cells. (B) IFN-γ expression was measured using theAlphaLISA detection kit (Perkin Elmer Waltham, Mass.) after 24 hours ofco-culture. No significant difference in T2 cell lysis was found betweenT cells in either 2-DG media or non-2-DG media at high peptideconcentration (1 μM) and slightly higher T2 cell lysis was found for Tcells at non-2-DG media than at lower peptide concentration (10 nM). Inaddition, 24 hour accumulated release of IFN-γ was higher for T cells innon-2-DG media cross all concentration of peptide tested. The lowerlevel of IFN-γ for T cells in 2-DG media at early timepoint (24 hours)might be due to lag. Further data (not shown here) suggested thatactivation in T cells from 2-DG media had a more prolonged activationthan in T cells from non-2-DG media, which could not be captured in ashort-term IFN-γ assay. (C) shows differentiation of T cells underdifferent media conditions. Culture media containing 2-DG was usedthroughout the entire process (10 days). T cells from the 2-DG mediawere less differentiated as suggested by the higher percentage ofcollected Tscm and Tcm subsets compared to the T cells from the non-2-DGmedia (59% vs 43%). This data suggests supplementing 2-DG in culturemedia for the entire process until harvest.

Example 3

This experiment compared CAR and TCR transduction of cells from anapheresed cell sample from a donor (Unenriched) with cells from the sameapheresed cell sample that was further enriched for T cells (Enriched).

On day 0, a fresh Leukopack (Hemacare, Northridge, Calif.), containing300 ml fresh enriched leukapheresis product collected from normalperipheral blood was sterile welded to a CD-600.1 Sepax Cell SeparationKit (GE) and processed under the Culture Wash-Pro program. TheLeukopack® contained leukocytes, erythrocytes, and platelets. The cellswere washed to remove plasma and apheresis buffers as described above inExample 1 and the nucleated cells were counted using NC-200™ AutomatedCell Counter and split equally into two transfer bags (Sample 1,Enriched T cells) and (Sample 2, Unenriched apheresed donor cells) usingthe Sepax C-Pro cell processing system Dilute program.

For Sample 1, “Enriched T cells”, the T cells were isolated from thewashed apheresed cells using an Easy Sep™ Human pan T negative selectionkit (Stemcell Technologies) according to the manufacturer'sinstructions. The enriched T cells were added to a gas permeable bag atcell density 4E6 nucleated cells/ml in 120 ml Optimizer complete mediacontaining 300 IU IL2. The gas permeable bag was previously coated with1.23 g/ml anti-CD3 antibody (Miltenyi Biotec) in PBS. Soluble anti-CD28antibody (Miltenyi Biotec) was added to the permeable bag at 1:100 (1μ/ml) dilution afterward for activation.

For Sample 2, “Unenriched apheresed donor cells”, same cell density (4E6nucleated cells/ml) was added into 120 ml Optimizer complete media(containing 300 IU/ml IL-2) into a gas permeable bag that was previouslycoated with anti-CD3 antibody as for Sample 1. Soluble anti-CD28antibody (Miltenyi Biotec) was added to the permeable bag at 1:100 (1μ/ml) dilution afterward for activation.

Day 1 The nucleated cells in each gas permeable bag were counted and theamount of lentiviral vector comprising a polynucleotide encoding a TCRcorresponding to a MOI of 1 (TCR) functional titer was added to one bag,a lentiviral vector comprising a polynucleotide encoding a CARcorresponding to a MOI of 40 (CAR) functional titer was added directlyto a second bag, and a lentiviral vector comprising a polynucleotideencoding green fluorescent protein (GFP) was added to the third gaspermeable bag. The bags were incubated at 37° C., 5% CO₂, overnight.

Days 2-6: On Day 2, an additional 120 ml OpTmizer complete mediumcontaining 300 IU/ml IL-2 was added into each gas permeable bag todilute the lentivirus.

Cells from each gas permeable were split into two bags on day 4, and onDay 6 were the bags were inoculated into equilibrated Xuri SP Perfbioreactor bags on Xuri W25 bioreactors (as described in Example 1),reaching a total volume of 800 ml culture media. The bioreactors wererocked at a rate of 6 rpm and at a 6° angle, at 37° C., 5% CO₂, gas flowrate of 0.1 L/min the entire time of expansion.

Days 7-13 or 14: On Day 7, the volume of culture media in thebioreactors was scaled up to 1 L. The cells were kept in bioreactoruntil Day 13 or 14, when they were harvested. During expansion,OpTimizer Complete media containing 300 IU/ml IL-2 was perfused at arate of 500 ml/day on Day 8, at a rate of 800 ml/day on Day 9, and at arate of 1 L/day from Day 10 until the time of harvest. Cell counts,viable cell density, cell viability, and metabolites were measured eachday.

The cells were harvested on Day 13 or 14. The bioreactor bags weresterile welded to a Sepax Pro using Culture Wash program and CT-600 CellProcessing kit (GE Healthcare). One wash cycle was performed usingsaline (Baxter) supplemented with 1% vol human serum albumin (HSA). Thewash was performed at 380×g for 5 min.

Viability and harvest recovery were measured. The cells wereconcentrated and further formulated at a 1:1 ratio with HyClone™Cryopreservation Media (GE Healthcare) supplemented with 5% human serumalbumin and cryopreserved by freezing to 100° C. using a Via Freeze (GEHealthcare) and storing in liquid nitrogen.

Results

Using unenriched apheresed donor cells as starting material improvedtransduction efficiency as seen in the level of surface expression andgenome integration compared with using enriched-T cells as startingmaterial. In addition, unenriched-cells from apheresis promoted aless-differentiated T cell phenotype. Two out of the three donors testedshowed a higher percentage of Tscm and Tcm upon harvest in both CD4⁺ andCD8⁺ T cell subsets. T cell purity was comparable between usingenriched- vs unenriched-T cells as starting material.

FIG. 14 shows transgene expression of T cells at harvest from enriched-and unenriched-cells. Three different lentiviral vectors delivering aCAR, a TCR, or a GFP were transduced into the enriched- andunenriched-donor cells. Transgene expression was detected via antibodystaining for the CAR, dextramer staining for the TCR, and fluorescentsignal for GFP, using flow cytometry. CAR, TCR, and GFP were higher inCD8 T cells from unenriched cells than from CD8⁺ T cells from enrichedprocess (A). Dextramer staining was CD8 receptor dependent, thus CD4 Tcells did not show expression of TCR (B). However, CAR and GFPexpression were both higher in CD4⁺ T cells from unenriched donor cellscompared to the enriched donor cells (B).

FIG. 15 shows the vector copy number of TCR in CD4⁺ and CD8⁺ T cells.Copies of WPRE and human albumin were measured via qPCR and vector copynumber of the TCR was calculated as a ratio of WPRE and human albumindivided by two. Vector copy number of the TCR was higher in both CD4⁺ Tcells and CD8⁺ T cells from unenriched donor cells than in the enricheddonor cells. It is found that the transgene integration was also higherin T cells from apheresis than in enriched T cells, indicated by highervector copy number.

FIG. 16 shows the percentage of leukocytes and leukocyte subsetsmeasured at the end of harvest. All cells were stained for leukocytes(CD45+). Minimal (<1%) B cells (CD19+), NK cells (CD56/16+), andmonocytes (CD14+) were detected in both unenriched and enriched donorcells. Overall the percentage of CD3⁺ cells (>99%) was comparablebetween the unenriched and enriched donor cells with the percentage of Tcells (CD3+CD56−) slightly lower in the enriched donor cells due toexpansion of NKT cells (CD3+CD56+).

FIG. 17 shows the T cell phenotype at harvest from the enriched- andunenriched-donor cells. This experiment was repeated three times with adifferent donor samples each time. Among the three donor samples tested,harvested T cells from two donors expressed higher percentage of memorystem cell markers (CD45RA+, CCR7+) and central memory markers (CD45RA−,CCR7+) from unenriched donor cells compared to those cells subject to Tcell enrichment. This difference was only significant in CD8⁺ T cellsbut not in CD4⁺ T cells.

FIG. 18 shows the growth curve and viability of cells in the bioreactorfrom enriched- and unenriched-donor cells. Growth (A) and viability (B)were comparable between the enriched- and unenriched-donor cells.

Example 4 this Example Provides a Closed Continuous Method for ProducingGenetically Engineered Autologous T Cells that Express a T Cell Receptor

On day 0, a fresh Leukopack (Hemacare, Northridge, Calif.), containing300 ml fresh enriched leukapheresis product collected from normalperipheral blood was sterile welded to a CD-600.1 Sepax Cell SeparationKit (GE) and processed under Culture Wash-Pro program. The Leukopackcontained leukocytes, erythrocytes, and platelets. The cells were washedin 1 L ClinMACS PBS/EDTA, 5 ml human serum albumin (Miltenyi Biotec, SanDiego, Calif.) to remove plasma and apheresis buffers using a Sepax CProequipped with a CS-600.1 Kit (GE Healthcare) according to manufacturer'sinstructions. Based on the initial white blood cell (WBC) countindicated on the donor information sheet accompanying the Leukopack®,the cells were eluted at a cell density of 150E6 WBC/ml with ˜50 mlOpTimizer complete media.

The nucleated cells in the washed leukapheresed harvest sample werecounted using NC200™ Automated Cell Counter and followed by determiningtotal viable cell count, viability, and immunophenotype of the washedcells.

A sample of the washed apheresed donor cells comprising 1.2E9 nucleatedcells was then transferred into a transfer bag (Charter Medicine) underthe Dilute program using the Sepax C-Pro processing system with the sameCD-600.1 Kit in ˜6.5 ml OpTimizer complete media containing 300 ml IL-2and was incubated with 7.5 ml ImmunoCult™ Human CD3/CD28/CD2 (2.5 μg/ml,Stemcell Technologies) at room temperature for 1 hour at roomtemperature to allow saturation of antibody binding.

A 2 L Xuri SP Perf Cellbag bioreactor (GE Healthcare) was connected to aXuri Cell Expansion System W25 and 300 ml OpTimizer complete mediacontaining 300 IU IL-2 was added and allowed to equilibrate at 37° C.,5% CO₂, gas flow rate of 0.1 L/min, and a rocking rate of 6 rpm at a 6°angle.

The transfer bag was sterile welded to the Xuri Cellbag feed-line andthe contents transferred into the bag by gravity. The cells were thenincubated overnight at 37° C., 5% CO₂, gas flow rate 0.1 L/min at arocking rate of 2 rpm at a 2° angle, to facilitate activation.

On Day 1, the nucleated cells in the bag were counted. An amount of alentiviral vector (comprising a polynucleotide encoding a TCR) thatcorresponded to a MOI of 1 functional titer was diluted in 10 ml ofOpTimizer complete media containing 300 IU/ml IL2 and was placed in atransfer bag. The transfer bag was sterile welded on to the Xuri Cellbagfeed-line and lentiviral vector was transferred into the bioreactor viagravity. The cells were incubated at 37° C., 5% CO₂, gas flow rate of0.1 L/min, and a rocking rate of 2 rpm at a 2° angle, for 20-24 hours.

On Day 2, about half the volume of the culture media in the bioreactorbag was exchanged for fresh OpTimizer complete media including 300 IU/mlIL2 using three 50 ml washouts. Cell counts were taken daily and viablecell density (VCD) and viability were determined using NC200, dissolvedoxygen, metabolites were also measured. Cell phenotypes were determinedfor all samples tested. The bags were maintained on the rockingbioreactors at 2 rpm at a 2° angle, 37° C., 5% CO₂, gas flow rate of 0.1L/min, for 24 hours.

On Days 3-10 the culture was maintained at 300 ml OpTimzer completemedia including 300 IU/ml IL2 by perfusion feeding at rate of one bagvolume per day, with a rocking rate of 2 rpm at a 2° angle, at 37° C.,5% CO₂, with gas flow rate of 0.1 L/min, until the cell density reached4e6 cells/ml.

At that point, the volume of the culture media was increased to 600 ml,perfusing at a rate of one bioreactor bag per day, with a rocking rateof 4 rpm at a 4° angle, at 37° C., 5% CO₂, with gas flow rate of 0.1L/min, until the cell density again reached 4e6 cells/ml.

At that point the volume of the culture media was increased to 1000 ml,perfusing at a rate of one bioreactor bag per day, with a rocking rateof 6 rpm at a 6° angle, at 37° C., 5% CO₂, with gas flow rate of 0.1L/min, until harvest on Day 10.

Total viable cell density, viability, glucose and lactate measurementswere taken each day. Phenotyping was determined on days 3, 5 and 7.

The cells were harvested on Day 10. The Xuri SP Per bioreactor bag wassterile welded to a Selfia 5200 Cell Processing System using theFlexCell program and CT-800.1 Cell Processing kit (GE Healthcare,). TheT cell were concentrated to ˜20 ml at a 75 ml/min flow rate. One washcycle was performed using 0.9% saline (Baxter, Deerfield, Ill.)supplemented with 1% vol human serum albumin (HSA). The wash wasperformed at 380×g for 5 min. The wash was performed at 380×g for 5 min.

The cells were then eluted at 2e8 cells/ml in saline supplemented with1% HSA and were further formulated at a 1:1 ratio with HyClone™Cryopreservation Media (GE Healthcare) supplemented with 5% human serumalbumin. Final cell product was then split into two freezing bags andseveral cryovials, which were finally frozen down in VIA Freeze™ Quadfreezer (GE) with a cooling rate of −1° C./min until the temperaturereached −80° C. After freezing, cells were transferred to liquidnitrogen for long-term storage.

Results

FIG. 19 shows the growth curve and viability of cells. The dip inviability is likely due to gradual die off of non-T cells.

FIG. 20 shows the percentage of leukocyte subsets at Day 0 and Day 10.The cells were stained with an antibody cocktail targeting B cells(CD19), Monocytes (CD14), T cells (CD3+, CD56/16−), NKT cells (CD3+,CD56/16+), NK cells (CD3−, CD56+), CD4 T cells (CD3+, CD4+), and CD8 Tcells (CD3+, CD8+) and analyzed by flow cytometry. CD3 cell purityreached 98% with total T cells at 98% and NKT cells at 10% by the timeof harvest (10 days).

FIG. 21 shows T cell differentiation during bioprocessing. T cellssubsets Tscm (CD45RA+, CD45RO−, CCR7+, CD95+), Tcm (CD45RA−, CD45RO+,CCR7+, CD95+), and Tem (CD45RA−, CD45RO+, CCR7−, CD95+) were stainedwith antibody cocktail and analyzed by flow cytometry at differenttimepoints. T cells differentiated similarly to that shown in inExamples 1 and 2 with around 70% Tem and around 30% Tscm+Tcm by the timeof harvest.

FIG. 22 shows the expression of transgene in harvested T cells at cellsurface level (A) and DNA level (B). Dextramer staining was performed toqualify surface expression of transgene on transduced and untransduced Tcells. Transgene expression was over 50% on transduced T cells (A). DNAwas extracted from harvested cells and 2 ng DNA was used for qPCRreactions targeting WPRE and human albumin. Vector copy number wascalculated as a ratio of copy number of WPRE and copy number of humanalbumins divided by 2. No detectable copy of transgene was found inuntransduced T cells and copy number of transgene in transduced T cellswas under 5, within the guideline on vector copy number implemented byFDA (B).

FIG. 23 shows the percentage of killing of target cells at differenteffector to target cells (E:T) ratios. Harvested T cells (transduced orun-transduced) were co-cultured with target cells for 24 hours at E:Tratio at 1:1, 1:5, and 1:10. Percentage of killing of target cells wascalculated by the percentage of cell death of target cells after 24hours of co-culture. Transduced T cells showed strong killing of targetcells (>70%) at all E:T ratio tested.

FIG. 24 shows IFN-gamma release of harvested cells. Harvested cells(transduced and untransduced) were co-cultured with target cells pulsedwith different concentrations of peptide for 24 hours. IFN-gamma in thesupernatant was measured via Alphalisa assay. Similar level of IFN-gammawas seen in Example 1 and 2.

The closed continuous autologous process generated over 26 billionengineered T cells rapidly (10 days), with high transgene expression(>50%) and CD3 cell purity (98%). The engineered T cells exhibitedremarkable killing potency against target cells at low effector totarget cells ratio (1:10) possibly resulting from robust IFN-gammarelease in response to target cells.

Example 5 Assessing Transduction Efficiency of a T Cell Expressing a TCRafter Stimulation with Different Reagents

On day 0, half of a fresh Leukopack (Hemacare, Northridge, Calif.),containing 150 ml leukapheresis product collected from normal peripheralblood was sterile welded to a CD-600.1 Sepax Cell Separation Kit (GE)and processed under Culture Wash-Pro program. The Leukopack® containedleukocytes, erythrocytes, and platelets. The cells were washed in 1 LClinMACS PBS/EDTA, 5 ml human serum albumin (Miltenyi Biotec, San Diego,Calif.) to remove plasma and apheresis buffers using a Sepax C-Pro CellSeparation System equipped with a CS-600.1 Kit (GE Healthcare),according to manufacturer's instructions.

The nucleated cells in the harvest sample were counted using NC-200™Automated Cell Counter (ChemoMetec) and followed by determining totalviable cell count, viability, and immunophenotype of the washed cells.

Starting populations of cells were prepared either by isolating pan Tcells from the washed cells donor cells using an Easy Sep™ Human pan Tnegative selection kit (Stemcell Technologies) according to themanufacturer's instructions or using the washed donor cells withoutfurther selection or enrichment, “unenriched unenriched cells”. Theenriched pan T cells and the unenriched cells were resuspended inOpTmizer complete media containing 300 IU/ml IL2 (StemcellTechnologies), unless otherwise noted, and added to transfer bags. Insome experiments IL-7, IL-15, IL-21, and/or TWS1119 (StemcellTechnologies) were used in the activation and expansion media.

Gas-permeable bags (PL07, Permalife, OriGen, Austin, Tex.) were preparedby coating with 1 ug/mL anti-CD3 (Clone OKT3 Miltenyi Biotech,Cambridge, Mass.) in PBS at 4° overnight and washed with PBS beforeadding cells. The following conditions were tested:

1) In one experiment, isolated pan T cells (2×10⁶ cells/mL) wereinoculated into anti-CD3 coated bags in OpTmizer complete mediacontaining 300 IU/ml IL2 (Stemcell Technologies) and 1 μg/ml anti-CD28(Miltenyi). For the titration of anti-CD3 or starting cell number,anti-CD3-coated plates (Corning) were used. The plates were coated withanti-CD3 at a titrated concentration from 0.0001-10 μg/ml. The cellswere activated for 20 hours and 48 hours. These cells were nottransduced. The levels of CD69, CD25, and 4-1BB on CD3+ T cells wasmeasured by flow cytometry at 20 and 48 hours (2 replicates percondition).

2) To compare the different activators, isolated pan T cells (2×10⁶cells/mL) were inoculated into anti-CD3 coated bag in OpTmizer completemedia containing 300 IU/ml IL2 (Stemcell Technologies) and 1 ug/mlanti-CD28 (Miltenyi). The following soluble activators were testedagainst the coated anti-CD3 condition. Immunocult anti-CD3/CD28(Stemcell Technologies, Cambridge, Mass.), and Immunocultanti-CD3/CD28/CD2 (Stemcell Technologies), each at 25 uL activator per 1mL of medium at 2E6 cells/mL, following the manufacturer's instructions.Dynabeads™ Human T-Activator CD3/CD28 (ThermoFisher) at a 1:1beads:cells ratio, following manufacturer's instructions, was tested inan uncoated bag. The cells/activators were incubated for 20 hours and 48hours.

3) Isolated pan T cells (2×10⁶ cells/mL) were inoculated into anti-CD3coated gas-permeable bags from above, in OpTmizer complete mediacontaining 300 IU/ml IL2 (Stemcell Technologies) and 1 ug/ml anti-CD28(Miltenyi). The following activators were tested: 1 ug/mL coatedanti-CD3 gas permeable bags from above with 1 ug/mL. Soluble anti-CD28(Miltenyi), soluble Immunocult anti-CD3/CD28/CD2 (StemcellTechnologies), at 25 uL activator per 1 mL of medium, Dynabeads™ HumanT-Activator CD3/CD28 (ThermoFisher) at a 1:1 beads:cells ratio, andsoluble MACS® GMP T Cell TransAct™ anti-CD3 and anti-CD28 conjugatedpolymeric nanomatrix, (Miltenyi Biotech) at a concentration of (1:17.5dilution), were tested in uncoated bags 48 hours after activation, thecells from the coated anti-CD3 bag were transferred to a new bag toremove the stimulation. Following activation, the cells were transduced,as described below. The cells/activators were incubated for 7 days.Engineered TCR expression was assessed by flow cytometry using adextramer recognizing the peptide-MHC class I complex, gated on CD8+ Tcells.

4) Washed leukaphereased cells (2×10⁶ cells/mL) were inoculated into theanti-CD3 coated gas-permeable bags from above in OpTmizer complete mediacontaining 300 IU/ml IL2 (Stemcell Technologies) and 1 ug/ml anti-CD28(Miltenyi). The following activators were tested: 1 ug/mL coatedanti-CD3 (Miltenyi) with 1 ug/mL soluble anti-CD28 (Miltenyi) andsoluble Immunocult anti-CD3/CD28/CD2 (Stemcell Technologies), at 25 uLactivator per 1 mL of medium. 48 hours after activation, the cells fromthe coated anti-CD3 bag were transferred to a new bag to remove thestimulation. Following activation, the cells were transduced, asdescribed below. The cells/activators were incubated for 7 days.Transduction efficiency was assessed by flow cytometry using GFPexpression, gating on either CD8+ or CD4+ T cells.

5) The effect of different cytokine cocktails. Fresh cells from theLeukopheresed material (WLPCs) were activated at 2×10⁶ cells/mL in gaspermeable bags with Immunocult anti-CD3/CD28/CD2 (Stemcell Technologies)in OpTmizer complete media containing 4 different cytokine cocktails: a)300 IU/ml IL2, b) 10 ng/mL IL-7 (Stemcell Technologies) and 10 ng/mLIL-15 (Stemcell Technologies), c) 10 uM TWS119 (Stemcell Technologies),10 ng/mL IL-7 (Stemcell Technologies), and 20 ng/mL IL-21 (StemcellTechnologies), or d) 10 ng/mL IL-7 (Stemcell Technologies) and 20 ng/mLIL-21 (Stemcell Technologies). Engineered TCR expression was assessed byflow cytometry using a dextramer specific to the TCR (Immudex), gatingon CD8+ T cells.

6) The effect of starting WLPC density for activation with solubleactivator. Cells were activated with Immunocult anti-CD3/CD28/CD2(Stemcell Technologies) in OpTmizer complete media containing 300 IU/mLIL-2 or 10 uM TWS119 (Stemcell Technologies) 10 ng/mL, IL-7 (StemcellTechnologies), and 20 ng/mL IL-21 (Stemcell Technologies at 4 differentdensities: a) 1×10⁶ cell/mL, b) 2×10⁶ cell/mL, c) 4×10⁶ cell/mL, or d)6×10⁶ cell/mL. Engineered TCR expression was assessed by flow cytometryusing a dextramer specific to the TCR (Immudex), gating on CD8+ T cells.

When transduced, 24 hours following activation, the cells in each bag(from experiments 3, 4, 5, and 6 above) were counted. An amount of alentivirus vector (comprising a polynucleotide encoding a TCR and GFP)that corresponded to a MOI of 1-2 functional titer was added to the bagand incubated overnight at 37° C., 5% CO₂.

The cells were expanded in static culture in the gas-permeable bags,keeping the cell density between 1-3 million cells/mL by splitting every2-3 days and transferring to larger gas-permeable bags as needed. Theexpansion was done for 6-9 days. Media with cytokines were replacedevery 2-3 days.

Cell counts were taken every 1-3 days.

Results

FIG. 25 shows the results of the titration of coated anti-CD3 (OKT3)antibody and pan T cell activation measured by CD69, CD25, and 4-1BB.Based on the stimulation of the T cells, 1 ug/ml anti-CD3 was chosen forcomparison with the soluble activators.

FIG. 26 shows the results of stimulation of pan T cells with varioussoluble activators versus coated anti-CD3 as measured by CD69, CD25, and4-1BB. Dynabeads (anti-CD3/CD28) showed the strongest stimulation,followed by the Immunocult anti-CD3/CD28/CD2 at the manufacturer'srecommended concentration of 25 μL/mL of cells in media. Interestingly,the soluble Immunocult anti-CD3/CD28 activator was comparable to coatedanti-CD3.

FIG. 27 shows the transduction efficiency of the engineered T cells wasdependent on stimulation signal. (A) Starting material: pan T cells.Dextramer staining of the engineered TCR on CD8+ T cells showedImmunocult anti-CD3/CD28/CD2 gave the highest transduction efficiency.(B) Starting material: apheresed cells: FACS for GFP expression intransduced CD4+ and CD8+ T cells showed Immunocult anti-CD3/CD28/CD2 wassuperior to coated anti-CD3.

FIG. 28 shows cell growth was similar between apheresed cells activatedcoated anti-CD3/soluble anti-CD28, Immunocult anti-CD3/CD28/CD2,TransAct, and Dynabeads for 9 days.

FIG. 29 shows culture conditions influenced the transduction efficiencyof the engineered T cells. Apheresed cells grown in culture mediacontaining IL-2, he combination of IL-7/15, of the combination ofIL-7/21 were similar in transduction efficiency. The cocktail containingIL-7/21/TWS119 improved transduction efficiency.

FIG. 30 shows starting cell density had an impact on transductionefficiency. Although there was no effect on the overall growth, higherstarting densities of apheresed cells that were activated withImmunocult anti-CD3/CD28/CD2 yielded better transduction efficiencieswhen culture media contained either IL-2 or a cocktail ofIL-7/21/TWS119.

This comparison of different T cell activators and cytokines using twodifferent starting cell populations, enriched pan T cells andleukapheresed cells. Activation strength did not appear to correlatewith better transduction efficiency, since Dynabeads anti-CD3/CD28(ThermoFisher) did not yield high transduction rates despite providingthe highest signal strength Immunocult anti-CD3/CD28/CD2 which had asignal strength higher than coated anti-CD3 but lower than Dynabeads,activated T cells with the highest expression of GFP and the engineeredTCR. Activating leukapheresed cells with Immunocult anti-CD3/CD28/CD2 inthe presence of different cytokine cocktails had an effect ontransduction efficiency, however when compared at different celldensities, transduction of cells activated and grow in culture mediacontaining IL-2 or IL-7/21/TWS119 were similar. Cells cultured in IL-2and IL-7/21/TWS119 were compared and it was found that theIL-7/21/TWS119 condition promoted elevated transduction efficiency.These results have important implications for the use of a solubleactivator, such as Immunocult anti-CD3/CD28/CD2, in a manufacturingprocess that leads to high numbers of functional engineered T cells forcell therapy applications.

Example 6 Optimum MOI for TCR Transduction

Previously frozen apheresed cells were thawed and activated withImmunocult CD3/CD28/CD2 (25 μl/ml of cells, Stemcell Technologies) for24 hours, as described above. On day 1 post activation, a lentiviralvector comprising a polynucleotide encoding a TRC was added to 1×10⁶cells at an increased multiplicity of infection (MOI) (A), equal to orgreater than a MOI of 1, or (B) decreasing MOIs of 2 or less. Cells wereassayed for TCR expression 7 days post-transduction. No observable MOIeffect was observed at MOIs greater than 1. Reported values are averagesof TCR expression in CD8+ T cells from 4 different donors. FIGS. 30A and7B show the MOI of 1 was optimal for lentiviral transduction inlarge-scale, closed bioprocess.

What is claimed is:
 1. A method for producing genetically engineeredautologous T cells expressing at least one protein of interest, themethod comprising inoculating a closed single use bioreactor bagcontaining culture media with apheresed donor cells and one or moresoluble T cell activators, wherein the bioreactor bag is part of arocking bioreactor platform, culturing the cells in the closed singleuse bioreactor bag continuously rocking at a rate of about 2 RPM,transducing the cells in the closed single use bioreactor bag with atleast one soluble viral vector comprising a polynucleotide which encodesthe protein of interest continuously rocking at a rate of about 2 RPM,and expanding the cells in the closed single use bioreactor bag at arocking rate of about 2 RPM and increasing the culture volume androcking rate as needed to maintain the culture until harvest.
 2. Themethod of claim 1, wherein the apheresed donor cells comprise cells fromperipheral blood.
 3. The method of claim 2, wherein the apheresed donorcells comprise nucleated and non-nucleated cells.
 4. The method of claim1, wherein the apheresed donor cells comprise leukocytes anderythrocytes.
 5. The method of claim 4, wherein the apheresed donorcells also comprise granulocytes and/or platelets.
 6. The method ofclaim 1, wherein the apheresis is leukapheresis.
 7. The method of claim1, wherein the apheresed donor cells are washed and resuspended in aculture media.
 8. The method of claim 1, wherein at least one T cellactivator is an anti CD3 antibody or binding fragments thereof.
 9. Themethod of claim 1, wherein the T cell activator comprises an anti CD3antibody and an anti CD28 antibody, or binding fragments thereof. 10.The method of claim 1, wherein the T cell activator comprises at leastan anti CD3 antibody, an anti CD28 antibody, and an anti CD2 antibody,or binding fragments thereof.
 11. The method of claim 1, wherein the Tcell activator comprises at least an anti-human CD3 monospecifictetrameric antibody complex, an anti-human CD28 monospecific tetramericantibody complex, and an anti-human CD2 monospecific tetrameric antibodycomplex.
 12. The method of claim 1, wherein the concentration of atleast one soluble T cell activator is at least 0.001 μg/ml to at least10 μg/ml.
 13. The method of claim 12, wherein the concentration of atleast one soluble T cell activator is at least 0.1 μg/ml to at least 5μg/ml.
 14. The method of claim 1, wherein at least one soluble T-cellactivator is bound to at least one donor cell at the time ofinoculation.
 15. The method of claim 1, wherein the apheresed donorcells are incubated with one or more soluble T cell activators prior toinoculating into the bioreactor bag.
 16. The method of claim 15, whereinthe incubation is for a sufficient time to allow for saturation ofbinding of one or more soluble T cell activator to the apheresed donorcells prior to inoculation.
 17. The method of claim 16, wherein theapheresed donor cells and one or more soluble T cell activators areincubated in a transfer bag.
 18. The method of claim 17, wherein thevolume of culture media in the transfer bag is about 5 ml to about 50ml.
 19. The method of claim 18, wherein the volume of culture media inthe transfer bag is about 5 ml to about 10 ml.
 20. The method of claim17, wherein the apheresed donor cells are incubated with one or more Tcell activators for at least 30 minutes or more.
 21. The method of claim20, wherein the apheresed donor cells are incubated with one or more Tcell activators for at least 1 hour.
 22. The method of claim 1, whereinthe number of nucleated cells within the apheresed donor cells is about1.0E9 to about 1.3E9.
 23. The method of claim 1, wherein the number ofnucleated cells within the apheresed donor cells is about 1.2E9.
 24. Themethod according to claim 1, wherein the bioreactor bag is inoculatedwith apheresed donor cells at a cell density of about 1E6 to about 5E6nucleated cells/ml.
 25. The method according to claim 24, wherein thebioreactor bag is inoculated with apheresed donor cells at a celldensity of about 2E6.
 26. The method according to claim 1, wherein thebioreactor bag contains at least 300 ml to at least 400 ml of culturemedia at inoculation.
 27. The method according to claim 26, wherein thebioreactor bag contains at least 300 ml of culture media at inoculation.28. The method according to claim 1, wherein the apheresed donor cellsare cultured in the bioreactor bag for about 12-24 hours.
 29. The methodof claim 1, wherein the culture media comprises at least one solublecytokine.
 30. The method according to claim 29, wherein the solublecytokine selected from IL-2, IL-7, IL-15, or IL-21.
 31. The methodaccording to claim 29, wherein at least one soluble cytokine is IL-2.32. The method according to claim 29, wherein the IL-2 is at aconcentration of about 250 IU/ml to about 350 IU/ml.
 33. The methodaccording to claim 32, wherein the IL-2 is at a concentration of about300 IU/ml.
 34. The method according to claim 29, wherein the solublecytokine is IL-7 in combination with IL-15 or IL-21.
 35. The methodaccording to claim 29, wherein the concentration of at least onecytokine is at least 5 ng/ml to at least 30 ng/ml.
 36. The methodaccording to claim 35, wherein the concentration of at least onecytokine is at least 10 ng/ml to at least 20 ng/ml.
 37. The methodaccording to claim 1, wherein the culture media also comprises a WNTpathway activator.
 38. The method according to claim 37, wherein the WNTpathway activator is TWS117.
 39. The method according to claim 37,wherein the culture media comprises a mixture of soluble TWS117, IL-7,and IL-21.
 40. The method of claim 1, wherein the culture media alsocomprises a soluble glycolysis inhibitor.
 41. The method of claim 40,wherein the soluble glycolysis inhibitor is 2-deoxy-D-glucose (2-DG).42. The method of claim 1, wherein the viral vector is a retroviralvector.
 43. The method of claim 1, wherein the viral vector is alentiviral vector.
 44. The method of claim 1, wherein the lentiviralvector is added at a MOI of 0.25-10.
 45. The method of claim 1, whereinthe lentiviral vector is added at a MOI of
 1. 46. The method of claim44, wherein the cells are transduced for about 20-24 hours.
 47. Themethod of claim 1, wherein following transduction, half of the culturemedia is removed from the bioreactor bag and replaced with an equalvolume of fresh culture media.
 48. The method of claim 47, wherein theculture is incubated for about 12-24 hours.
 49. The method of claim 1,wherein during expansion, fresh culture media is added to the bioreactorby fed batch/perfusion feeding and/or by perfusion.
 50. The method ofclaim 1, wherein during expansion the culture is perfused at a rate isone bioreactor bag volume per day.
 51. The method of claim 1, as thecells are expanded the volume of the culture media in bioreactor isincrementally increased to 1 liter during expansion.
 52. The method ofclaim 1, as the cells are expanded the volume of the culture media isincrementally increased to maintain a cell density of at least 2E6nucleated cells/ml.
 53. The method of claim 1, as the cells are expandedthe volume of the culture media in bioreactor is incrementally increasedto 1 liter during expansion to maintain a cell density of at least 4E6nucleated cells/ml.
 54. The method of claim 1, as the cells are expandedthe rocking rate is incrementally increased to 6 RPM.
 55. The method ofclaim 1, at the start of expansion the volume of culture media in thesingle use closed bioreactor bag is 300 ml rocking at a rate of 2 rpm ata 2° angle.
 56. The method of claim 1, wherein the culture in the singleuse closed bioreactor bag is maintained at about 80-100% O₂.
 57. Themethod of claim 1, wherein the cells are expanded for 7 to 14 days. 58.The method of claim 1, wherein the culture, transduction, and/orexpansion steps are performed at 34-37° C.
 59. The method of claim 1,wherein the protein of interest is a cell surface receptor.
 60. Themethod of claim 59, wherein the cell surface receptor a T cell receptor,or chimeric antigen receptor.
 61. The method of claim 60, wherein thecell surface receptor recognizes an antigenic target associated with atarget cell.
 62. The method of claim 61, wherein the target cell is acancer cell.
 63. The method of claim 1, wherein the geneticallyengineered autologous T cells are used to treat an indication in apatient in need.
 64. A pharmaceutical composition comprising thegenetically engineered autologous T cells of claim
 1. 65. A method oftreating an indication in a patient in need, comprising administering tothe patient the pharmaceutical composition of claim
 64. 66. A method forincreasing the transgene expression in genetically engineered autologousT cells expressing a protein of interest, the method comprisinginoculating a closed single use bioreactor bag containing culture mediawith apheresed donor cells and one or more soluble T cell activators,wherein at least one soluble T-cell activator is bound to at least onedonor cell at the time of inoculation and the bioreactor bag is part ofa rocking bioreactor platform, culturing the cells in the closed singleuse bioreactor bag continuously rocking at a rate of about 2 RPM,transducing the cells in the closed single use bioreactor bag with atleast one soluble viral vector comprising a polynucleotide which encodesthe protein of interest continuously rocking at a rate of about 2 RPM,and expanding the cells in the closed single use bioreactor bag at arocking rate of about 2 RPM and increasing the culture volume androcking the rate as needed to maintain the culture until harvest,wherein the transgene expression is greater than the transgeneexpression of genetically engineered autologous T cells derived from anenriched population of T cells from the same apheresed donor cells andexpressing the same protein of interest.
 67. A method of treating apatient with genetically engineered autologous T cells expressing aprotein of interest comprising, incubating apheresed cells from thepatient with one or more T cell activators selected from the groupconsisting of an anti CD3 antibody, an anti CD2 antibody, and an antiCD28 antibody or binding fragments thereof, to allow for saturation ofantibody binding, inoculating a closed single use bioreactor bagcontaining culture media with the apheresed cells, wherein thebioreactor bag is part of a rocking bioreactor platform, culturing thecells in the closed single use bioreactor bag continuously rocking at arate of about 2 RPM, transducing the cells in the closed single usebioreactor bag with at least one soluble viral vector comprising apolynucleotide which encodes the protein of interest continuouslyrocking at a rate of about 2 RPM, and expanding the cells in the closedsingle use bioreactor bag at a rocking rate of about 2 RPM, increasingthe culture volume and rocking the rate as needed to maintain theculture at a desired cell density until harvest. harvesting andformulating the cells for cryopreservation, freezing the cells andstoring until needed for administering to the patient, thawing andresuspending the cells in a suitable media for infusion, andreintroducing a pharmaceutically effective amount of the geneticallyengineered autologous T cells expressing the protein of interest intothe patient.